Production of polypeptides

ABSTRACT

An improved system for large scale production of proteins and/or polypeptides in cell culture, particularly in media characterized by one or more of: i) a cumulative amino acid concentration greater than about 70 mM; ii) a molar cumulative glutamine to cumulative asparagine ratio of less than about 2; iii) a molar cumulative glutamine to cumulative total amino acid ratio of less than about 0.2; iv) a molar cumulative inorganic ion to cumulative total amino acid ratio between about 0.4 to 1; or v) a combined cumulative glutamine and cumulative asparagine concentration between about 16 and 36 mM, is provided. The use of such a system allows high levels of protein production and lessens accumulation of certain undesirable factors such as ammonium and/or lactate. Additionally, culture methods including a temperature shift, typically including a decrease in temperature when the culture has reached about 20-80% of it maximal cell density, are provided. Alternatively or additionally, the present invention provides methods such that, after reaching a peak, lactate and/or ammonium levels in the culture decrease over time.

RELATED APPLICATIONS

This application claims priority to Provisional Patent Application Nos.60/605,097, 60/604,941, and 60/605,074, each of which was filed Aug. 27,2004, and each of which is incorporated herein by reference in itsentirety

BACKGROUND OF THE INVENTION

Proteins and polypeptides have become increasingly important astherapeutic agents. In most cases, therapeutic proteins and polypeptidesare produced in cell culture, from cells that have been engineeredand/or selected to produce unusually high levels of the particularprotein or polypeptide of interest. Control and optimization of cellculture conditions is critically important for successful commercialproduction of proteins and polypeptides.

Many proteins and polypeptides produced in cell culture are made in abatch or fed-batch process, in which cells are cultured for a period oftime, and then the culture is terminated and the produced protein orpolypeptide is isolated. The ultimate amount and quality of protein orpolypeptide produced can be dramatically affected by the conditions ofthe cell culture. For example, traditional batch and fed-batch cultureprocesses often result in production of metabolic waste products thathave detrimental effects on cell growth, viability, and production orstability of the protein or polypeptide of interest. While efforts havebeen made to improve production of proteins and polypeptides in batchand fed-batch culture processes, there remains a need for additionalimprovements.

Additionally, significant effort has been invested in the development ofdefined media (i.e., media assembled from known individual componentsand lacking serum or other animal byproducts) for use in culturingcells, particularly mammalian cells. Cell growth characteristics can bevery different in defined media as contrasted with serum-derived media.There is a particular need for the development of improved systems forproducing proteins and polypeptides by cell culture in defined media.

SUMMARY OF THE INVENTION

The present invention provides an improved system for large scaleproduction of proteins and/or polypeptides in cell culture. For example,the present invention provides commercial scale (e.g., 500 L or more)culture methods that utilize a medium characterized by one or more of:i) a cumulative amino acid amount per unit volume greater than about 70mM; ii) a molar cumulative glutamine to cumulative asparagine ratio ofless than about 2; iii) a molar cumulative glutamine to cumulative totalamino acid ratio of less than about 0.2; iv) a molar cumulativeinorganic ion to cumulative total amino acid ratio between about 0.4 to1; or v) a combined cumulative amount of glutamine and asparagineconcentration per unit volume greater than about 16 mM. One of ordinaryskill in the art will understand that “cumulative”, as used above,refers to the total amount of a particular component or components addedover the course of the cell culture, including components added at thebeginning of the culture and subsequently added components. In certainpreferred embodiments of the invention, it is desirable to minimize“feeds” of the culture over time, so that it is desirable to maximizeamounts present initially. Of course, medium components are metabolizedduring culture so that cultures with the same cumulative amounts ofgiven components will have different absolute levels if those componentsare added at different times (e.g., all present initially vs. some addedby feeds).

According to the present invention, use of such a medium allows highlevels of protein production and lessens accumulation of certainundesirable factors such as ammonium and/or lactate.

One of ordinary skill in the art will understand that the mediaformulations of the present invention encompass both defined andnon-defined media. In certain preferred embodiments of the presentinvention, the culture medium is a defined medium in which thecomposition of the medium is known and controlled.

In certain preferred embodiments of the present invention, the culturemethods include changing the culture from a first set of cultureconditions to a second set of culture conditions so that a metabolicshift of the cells is achieved. In some embodiments, this change isperformed when the culture has reached about 20-80% of its maximal celldensity. In some embodiments, the change involves changing thetemperature (or temperature range) at which the culture is maintained.Alternatively or additionally, the present invention provides methodsadjusted so that, after reaching a peak, lactate and/or ammonium levelsin the culture decrease over time. In other embodiments, the shiftinvolves shifting the pH, osmolarlity or level of chemical inductants,such as alkanoic acids or their salts.

Cell cultures of the present invention may optionally be supplementedwith nutrients and/or other medium components including hormones and/orother growth factors, particular ions (such as sodium, chloride,calcium, magnesium, and phosphate), buffers, vitamins, nucleosides ornucleotides, trace elements (inorganic compounds usually present at verylow final concentrations), amino acids, lipids, or glucose or otherenergy source. In certain embodiments of the present invention, it maybe beneficial to supplement the media with chemical inductants such ashexamethylene-bis(acetamide) (“HMBA”) and sodium butyrate (“NaB”). Theseoptional supplements may be added at the beginning of the culture or maybe added at a later point in order to replenish depleted nutrients orfor another reason. In general, it is desirable to select the initialmedium composition to minimize supplementation in accordance with thepresent invention.

Various culture conditions may be monitored in accordance with thepresent invention, including pH, cell density, cell viability, lactatelevels, ammonium levels, osmolarity, or titer of the expressedpolypeptide or protein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a comparison of Medium 1 and Medium 2 in shake flasks usinganti-GDF-8 cells.

FIG. 2 shows cell growth and viability of anti-GDF-8 cells in Medium 1.

FIG. 3 shows cell growth of anti-GDF-8 cell cultures in control and noglutamine feed culture conditions.

FIG. 4 shows cell viability of anti-GDF-8 cell cultures in control andno glutamine feed culture conditions.

FIG. 5 shows ammonium levels of anti-GDF-8 cell cultures in control andno glutamine feed culture conditions.

FIG. 6 shows lactate levels of anti-GDF-8 cell cultures in control andno glutamine feed culture conditions.

FIG. 7 shows anti-GDF-8 titer in control and no glutamine feed cultureconditions.

FIG. 8 shows cell density of anti-GDF-8 cell cultures in control andglutamine-starved feed culture conditions.

FIG. 9 shows cell viability of anti-GDF-8 cell cultures in control andglutamine-starved feed culture conditions.

FIG. 10 shows ammonium levels of anti-GDF-8 cell cultures in control andglutamine-starved culture conditions.

FIG. 11 shows lactate levels of anti-GDF-8 cell cultures in control andglutamine-starved culture conditions.

FIG. 12 shows anti-GDF-8 titer in control and glutamine-starved cultureconditions.

FIG. 13 shows iron dose response of anti-GDF-8 cells in Medium 1 andMedium 2.

FIG. 14 shows cell density of Glutamate and Glutamine fed cultures.

FIG. 15 shows cell viability of Glutamate and Glutamine fed cultures.

FIG. 16 shows anti-Lewis Y titer in Glutamate and Glutamine fedcultures.

FIG. 17 shows lactate levels in Glutamate and Glutamine fed cultures.

FIG. 18 shows ammonium levels in Glutamate and Glutamine fed cultures.

FIG. 19 shows osmolarity of Glutamate and Glutamine fed cultures.

FIG. 20 shows cell density of anti-Lewis Y cells. Each plot is theaverage of two shake flasks grown using the same conditions.

FIG. 21 shows cell viability of anti-Lewis Y cells. Each plot is theaverage of two shake flasks grown using the same conditions.

FIG. 22 shows average titer of anti-Lewis Y culture. Each plot is theaverage of two shake flasks grown using the same conditions.

FIG. 23 shows ammonium levels of anti-Lewis Y cells. Each plot is theaverage of two shake flasks grown using the same conditions.

FIG. 24 shows an impeller jump used in fed-batch cultures.

FIG. 25 shows cell growth of anti-GDF-8 cells under various experimentalconditions.

FIG. 26 shows viability of anti-GDF-8 cells under various experimentalconditions.

FIG. 27 shows anti-GDF-8 titer under various experimental conditions.

FIG. 28 shows lactate levels of anti-GDF-8 cultures under variousexperimental conditions.

FIG. 29 shows ammonium levels of anti-GDF-8 cultures under variousexperimental conditions.

FIG. 30 shows cell growth of anti-GDF-8 cells under various experimentalconditions.

FIG. 31 shows anti-GDF-8 titer under various experimental conditions.

FIG. 32 shows lactate levels of anti-GDF-8 cultures under variousexperimental conditions.

FIG. 33 shows ammonium levels of anti-GDF-8 cultures under variousexperimental conditions.

FIG. 34 shows cell growth of anti-GDF-8 cells in modified Medium 9containing various levels of glutamine and asparagine.

FIG. 35 shows cell viability of anti-GDF-8 cells in modified Medium 9containing various levels of glutamine and asparagine.

FIG. 36 shows lactate levels of anti-GDF-8 cultures in modified Medium 9containing various levels of glutamine and asparagine.

FIG. 37 shows ammonium levels of anti-GDF-8 cultures in modified Medium9 containing various levels of glutamine and asparagine.

FIG. 38 shows glutamine levels of anti-GDF-8 cultures in modified Medium9 containing various levels of glutamine and asparagine.

FIG. 39 shows anti-GDF-8 titer in modified Medium 9 containing variouslevels of glutamine and asparagine.

FIG. 40 shows osmolarity of anti-GDF-8 cultures in modified Medium 9containing various levels of glutamine and asparagine.

FIG. 41 shows cell growth of anti-GDF-8 cells in media containingvarious levels of asparagine and cysteine.

FIG. 42 shows lactate levels of anti-GDF-8 cultures in media containingvarious levels of asparagine and cysteine.

FIG. 43 shows ammonium levels of anti-GDF-8 cultures in media containingvarious levels of asparagine and cysteine.

FIG. 44 shows glutamine levels of anti-GDF-8 cultures in mediacontaining various levels of asparagine and cysteine.

FIG. 45 shows glutamate levels of anti-GDF-8 cultures in mediacontaining various levels of asparagine and cysteine.

FIG. 46 shows anti-GDF-8 titer in media containing various levels ofasparagine and cysteine.

FIG. 47 shows osmolarity of anti-GDF-8 cultures in media containingvarious levels of asparagine and cysteine.

FIG. 48 shows cell growth of anti-GDF-8 cells in media containingvarious levels of amino acids and vitamins.

FIG. 49 shows lactate levels of anti-GDF-8 cultures in media containingvarious levels of amino acids and vitamins.

FIG. 50 shows ammonium levels of anti-GDF-8 cultures in media containingvarious levels of amino acids and vitamins.

FIG. 51 shows glutamine levels of anti-GDF-8 cultures in mediacontaining various levels of amino acids and vitamins.

FIG. 52 shows anti-GDF-8 titer in media containing various levels ofamino acids and vitamins.

FIG. 53 shows cell growth of anti-GDF-8 cells in media containingvarious levels of vitamins, trace elements E and iron.

FIG. 54 shows lactate levels of anti-GDF-8 cultures in media containingvarious levels of vitamins, trace elements E and iron.

FIG. 55 shows ammonium levels of anti-GDF-8 cultures in media containingvarious levels of vitamins, trace elements E and iron.

FIG. 56 shows anti-GDF-8 titer in media containing various levels ofvitamins, trace elements E and iron.

FIG. 57 shows cell growth of anti-GDF-8 cells in Mediums 1, 3 and 9.

FIG. 58 shows anti-GDF-8 titer in Medium 1, 3 and 9.

FIG. 59 shows extrapolated anti-GDF-8 titers for various levels ofglutamine alone and total combined glutamine and asparagine.

FIG. 60 shows cell growth of anti-ABeta cells under various mediaconditions tested.

FIG. 61 shows cell viability of anti-ABeta cells under various mediaconditions tested.

FIG. 62 shows lactate levels of anti-ABeta cultures under various mediaconditions tested.

FIG. 63 shows ammonium levels of anti-ABeta cultures under various mediaconditions tested.

FIG. 64 shows anti-ABeta titer in various media conditions tested.

FIG. 65 shows osmolarity of anti-ABeta cultures under various mediaconditions tested.

FIG. 66 shows cell growth of cells expressing TNFR-Ig under variousexperimental conditions.

FIG. 67 shows viability of cells expressing TNFR-Ig under variousexperimental conditions.

FIG. 68 shows residual glucose in cultures of cells expressing TNFR-Igunder various experimental conditions.

FIG. 69 shows glutamine levels in cultures of cells expressing TNFR-Igunder various experimental conditions.

FIG. 70 shows lactate concentration in cultures of cells expressingTNFR-Ig under various experimental conditions.

FIG. 71 shows ammonium levels in cultures of cells expressing TNFR-Igunder various experimental conditions.

FIG. 72 shows TNFR-Ig relative titer under various experimentalconditions.

FIG. 73 shows cell densities of anti-GDF-8 cells grown in 6000 L and 1 Lbioreactors.

FIG. 74 shows titers of anti-GDF-8 cells grown in 6000 L and 1 Lbioreactors.

FIG. 75 shows lactate levels of anti-GDF-8 cells grown in 6000 L and 1 Lbioreactors.

FIG. 76 shows ammonium levels of anti-GDF-8 cells grown in 6000 L and 1L bioreactors.

DEFINITIONS

“About”, “Approximately”: As used herein, the terms “about” and“approximately”, as applied to one or more particular cell cultureconditions, refer to a range of values that are similar to the statedreference value for that culture condition or conditions. In certainembodiments, the term “about” refers to a range of values that fallwithin 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, 1 percent or less of the stated reference value for that culturecondition or conditions.

“Amino acid”: The term “amino acid” as used herein refers to any of thetwenty naturally occurring amino acids that are normally used in theformation of polypeptides, or analogs or derivatives of those aminoacids. Amino acids of the present invention are provided in medium tocell cultures. The amino acids provided in the medium may be provided assalts or in hydrate form.

“Antibody”: The term “antibody” as used herein refers to animmunoglobulin molecule or an immunologically active portion of animmunoglobulin molecule, such as a Fab or F(ab′)₂ fragment, thatcontains one or more antigen binding sites which specifically bind(immunoreact with) an antigen. The terms “monoclonal antibodies” and“monoclonal antibody composition”, as used herein, refer to a clonalpopulation of antibody molecules that contain only one species of anantigen binding site capable of immunoreacting with a particular epitopeof an antigen, whereas the terms “polyclonal antibodies” and “polyclonalantibody composition” refer to a population of antibody molecules thatcontain multiple species of antigen binding sites capable of interactingwith a particular antigen. The definition of monoclonal antibodiesincludes both clonal molecules derived by traditional technologies aswell as molecules of defined sequence derived by manipulation ormutation of specific residues, for example, humanized antibodies.

“Batch culture”: The term “batch culture” as used herein refers to amethod of culturing cells in which all the components that willultimately be used in culturing the cells, including the medium (seedefinition of “medium” below) as well as the cells themselves, areprovided at the beginning of the culturing process. A batch culture istypically stopped at some point and the cells and/or components in themedium are harvested and optionally purified.

“Bioreactor”: The term “bioreactor” as used herein refers to any vesselused for the growth of a mammalian cell culture. The bioreactor can beof any size so long as it is useful for the culturing of mammaliancells. Typically, the bioreactor will be at least 1 liter and may be 10,100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,0000 liters or more,or any volume in between. The internal conditions of the bioreactor,including, but not limited to pH and temperature, are typicallycontrolled during the culturing period. The bioreactor can be composedof any material that is suitable for holding mammalian cell culturessuspended in media under the culture conditions of the presentinvention, including glass, plastic or metal. The term “productionbioreactor” as used herein refers to the final bioreactor used in theproduction of the polypeptide or protein of interest. The volume of thelarge-scale cell culture production bioreactor is typically at least 500liters and may be 1000, 2500, 5000, 8000, 10,000, 12,0000 liters ormore, or any volume in between. One of ordinary skill in the art will beaware of and will be able to choose suitable bioreactors for use inpracticing the present invention.

“Cell density”: The term “cell density” as used herein refers to thatnumber of cells present in a given volume of medium.

“Cell viability”: The term “cell viability” as used herein refers to theability of cells in culture to survive under a given set of cultureconditions or experimental variations. The term as used herein alsorefers to that portion of cells which are alive at a particular time inrelation to the total number of cells, living and dead, in the cultureat that time.

“Culture”, “Cell culture” and “Mammalian cell culture”: These terms asused herein refer to a mammalian cell population that is suspended in amedium (see definition of “medium” below) under conditions suitable tosurvival and/or growth of the cell population. As will be clear to thoseof ordinary skill in the art, these terms as used herein may refer tothe combination comprising the mammalian cell population and the mediumin which the population is suspended.

“Fed-batch culture”: The term “fed-batch culture” as used herein refersto a method of culturing cells in which additional components areprovided to the culture at some time subsequent to the beginning of theculture process. The provided components typically comprise nutritionalsupplements for the cells which have been depleted during the culturingprocess. A fed-batch culture is typically stopped at some point and thecells and/or components in the medium are harvested and optionallypurified.

“Fragment”: The term “fragment” as used herein refers to polypeptidesand is defined as any discrete portion of a given polypeptide that isunique to or characteristic of that polypeptide. The term as used hereinalso refers to any discrete portion of a given polypeptide that retainsat least a fraction of the activity of the full-length polypeptide.Preferably the fraction of activity retained is at least 10% of theactivity of the full-length polypeptide. More preferably the fraction ofactivity retained is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%of the activity of the full-length polypeptide. More preferably stillthe fraction of activity retained is at least 95%, 96%, 97%, 98% or 99%of the activity of the full-length polypeptide. Most preferably, thefraction of activity retained is 100% of the activity of the full-lengthpolypeptide. The term as used herein also refers to any portion of agiven polypeptide that includes at least an established sequence elementfound in the full-length polypeptide. Preferably, the sequence elementspans at least 4-5, more preferably at least about 10, 15, 20, 25, 30,35, 40, 45, 50 or more amino acids of the full-length polypeptide.

“Gene”: The term “gene” as used herein refers to any nucleotidesequence, DNA or RNA, at least some portion of which encodes a discretefinal product, typically, but not limited to, a polypeptide, whichfunctions in some aspect of cellular metabolism or development. The termis not meant to refer only to the coding sequence that encodes thepolypeptide or other discrete final product, but may also encompassregions preceding and following the coding sequence that modulate thebasal level of expression (see definition of “genetic control element”below), as well as intervening sequences (“introns”) between individualcoding segments (“exons”).

“Genetic control element”: The term “genetic control element” as usedherein refers to any sequence element that modulates the expression of agene to which it is operably linked. Genetic control elements mayfunction by either increasing or decreasing the expression levels andmay be located before, within or after the coding sequence. Geneticcontrol elements may act at any stage of gene expression by regulating,for example, initiation, elongation or termination of transcription,mRNA splicing, mRNA editing, mRNA stability, mRNA localization withinthe cell, initiation, elongation or termination of translation, or anyother stage of gene expression. Genetic control elements may functionindividually or in combination with one another.

“Hybridoma”: The term “hybridoma” as used herein refers to a cellcreated by fusion of an immortalized cell derived from an immunologicsource and an antibody-producing cell. The resulting hybridoma is animmortalized cell that produces antibodies. The individual cells used tocreate the hybridoma can be from any mammalian source, including, butnot limited to, rat, pig, rabbit, sheep, pig, goat, and human. The termalso encompasses trioma cell lines, which result when progeny ofheterohybrid myeloma fusions, which are the product of a fusion betweenhuman cells and a murine myeloma cell line, are subsequently fused witha plasma cell. Furthermore, the term is meant to include anyimmortalized hybrid cell line that produces antibodies such as, forexample, quadromas (See, e.g., Milstein et al., Nature, 537:3053(1983)).

“Integrated Viable Cell Density”: The term “integrated viable celldensity” as used herein refers to the average density of viable cellsover the course of the culture multiplied by the amount of time theculture has run. Assuming the amount of polypeptide and/or proteinproduced is proportional to the number of viable cells present over thecourse of the culture, integrated viable cell density is a useful toolfor estimating the amount of polypeptide and/or protein produced overthe course of the culture.

“Medium”, “Cell culture medium”, “Culture medium”: These terms as usedherein refer to a solution containing nutrients which nourish growingmammalian cells. Typically, these solutions provide essential andnon-essential amino acids, vitamins, energy sources, lipids, and traceelements required by the cell for minimal growth and/or survival. Thesolution may also contain components that enhance growth and/or survivalabove the minimal rate, including hormones and growth factors. Thesolution is preferably formulated to a pH and salt concentration optimalfor cell survival and proliferation. The medium may also be a “definedmedia”—a serum-free media that contains no proteins, hydrolysates orcomponents of unknown composition. Defined media are free ofanimal-derived components and all components have a known chemicalstructure.

“Metabolic waste product”: The term “metabolic waste product” as usedherein refers to compounds produced by the cell culture as a result ofnormal or non-normal metabolic processes that are in some waydetrimental to the cell culture, particularly in relation to theexpression or activity of a desired recombinant polypeptide or protein.For example, the metabolic waste products may be detrimental to thegrowth or viability of the cell culture, may decrease the amount ofrecombinant polypeptide or protein produced, may alter the folding,stability, glycoslyation or other post-translational modification of theexpressed polypeptide or protein, or may be detrimental to the cellsand/or expression or activity of the recombinant polypeptide or proteinin any number of other ways. Exemplary metabolic waste products includelactate, which is produced as a result of glucose metabolism, andammonium, which is produced as a result of glutamine metabolism. Onegoal of the present invention is to slow production of, reduce or eveneliminate metabolic waste products in mammalian cell cultures.

“Osmolarity” and “Osmolality”: “Osmolality” is a measure of the osmoticpressure of dissolved solute particles in an aqueous solution. Thesolute particles include both ions and non-ionized molecules. Osmolalityis expressed as the concentration of osmotically active particles (i.e.,osmoles) dissolved in 1 kg of solution (1 mOsm/kg H₂O at 38° C. isequivalent to an osmotic pressure of 19 mm Hg). “Osmolarity,” bycontrast, refers to the number of solute particles dissolved in 1 literof solution. When used herein, the abbreviation “mOsm” means“milliosmoles/kg solution”.

“Perfusion culture”: The term “perfusion culture” as used herein refersto a method of culturing cells in which additional components areprovided continuously or semi-continuously to the culture subsequent tothe beginning of the culture process. The provided components typicallycomprise nutritional supplements for the cells which have been depletedduring the culturing process. A portion of the cells and/or componentsin the medium are typically harvested on a continuous or semi-continuousbasis and are optionally purified.

“Polypeptide”: The term “polypeptide” as used herein refers a sequentialchain of amino acids linked together via peptide bonds. The term is usedto refer to an amino acid chain of any length, but one of ordinary skillin the art will understand that the term is not limited to lengthychains and can refer to a minimal chain comprising two amino acidslinked together via a peptide bond.

“Protein”: The term “protein” as used herein refers to one or morepolypeptides that function as a discrete unit. If a single polypeptideis the discrete functioning unit and does require permanent physicalassociation with other polypeptides in order to form the discretefunctioning unit, the terms “polypeptide” and “protein” as used hereinare used interchangeably. If discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” as used herein refers to the multiple polypeptidesthat are physically coupled and function together as the discrete unit.

“Recombinantly expressed polypeptide” and “Recombinant polypeptide”:These terms as used herein refer to a polypeptide expressed from amammalian host cell that has been genetically engineered to express thatpolypeptide. The recombinantly expressed polypeptide can be identical orsimilar to polypeptides that are normally expressed in the mammalianhost cell. The recombinantly expressed polypeptide can also foreign tothe host cell, i.e. heterologous to peptides normally expressed in themammalian host cell. Alternatively, the recombinantly expressedpolypeptide can be chimeric in that portions of the polypeptide containamino acid sequences that are identical or similar to polypeptidesnormally expressed in the mammalian host cell, while other portions areforeign to the host cell.

“Seeding”: The term “seeding” as used herein refers to the process ofproviding a cell culture to a bioreactor or another vessel. The cellsmay have been propagated previously in another bioreactor or vessel.Alternatively, the cells may have been frozen and thawed immediatelyprior to providing them to the bioreactor or vessel. The term refers toany number of cells, including a single cell.

“Titer”: The term “titer” as used herein refers to the total amount ofrecombinantly expressed polypeptide or protein produced by a mammaliancell culture divided by a given amount of medium volume. Titer istypically expressed in units of milligrams of polypeptide or protein permilliliter of medium.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The present invention provides improved systems for the production ofproteins and/or polypeptides by cell culture. In particular, theinvention provides systems that minimize production of one or moremetabolic products detrimental to cell growth, viability, and/or proteinproduction or quality. In a preferred embodiment of the presentinvention, the cell culture is a batch or fed-batch culture. Othercertain preferred embodiments of the invention are discussed in detailbelow. Those of ordinary skill in the art will understand, however, thatvarious modifications to these preferred embodiments are within thescope of the appended claims. It is the claims and equivalents thereofthat define the scope of the present invention, which is not and shouldnot be limited to or by this description of certain preferredembodiments.

Polypeptides

Any polypeptide that is expressible in a host cell may be produced inaccordance with the present invention. The polypeptide may be expressedfrom a gene that is endogenous to the host cell, or from a gene that isintroduced into the host cell through genetic engineering. Thepolypeptide may be one that occurs in nature, or may alternatively havea sequence that was engineered or selected by the hand of man. Anengineered polypeptide may be assembled from other polypeptide segmentsthat individually occur in nature, or may include one or more segmentsthat are not naturally occurring.

Polypeptides that may desirably be expressed in accordance with thepresent invention will often be selected on the basis of an interestingbiological or chemical activity. For example, the present invention maybe employed to express any pharmaceutically or commercially relevantenzyme, receptor, antibody, hormone, regulatory factor, antigen, bindingagent, etc.

Antibodies

Given the large number of antibodies currently in use or underinvestigation as pharmaceutical or other commercial agents, productionof antibodies is of particular interest in accordance with the presentinvention. Antibodies are proteins that have the ability to specificallybind a particular antigen. Any antibody that can be expressed in a hostcell may be used in accordance with the present invention. In apreferred embodiment, the antibody to be expressed is a monoclonalantibody.

In another preferred embodiment, the monoclonal antibody is a chimericantibody. A chimeric antibody contains amino acid fragments that arederived from more than one organism. Chimeric antibody molecules caninclude, for example, an antigen binding domain from an antibody of amouse, rat, or other species, with human constant regions. A variety ofapproaches for making chimeric antibodies have been described. See e.g.,Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81, 6851 (1985); Takedaet al., Nature 314, 452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567;Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP 171496; European Patent Publication 0173494, UnitedKingdom Patent GB 2177096B.

In another preferred embodiment, the monoclonal antibody is a humanantibody derived, e.g., through the use of ribosome-display orphage-display libraries (see, e.g., Winter et al., U.S. Pat. No.6,291,159 and Kawasaki, U.S. Pat. No. 5,658,754) or the use ofxenographic species in which the native antibody genes are inactivatedand functionally replaced with human antibody genes, while leavingintact the other components of the native immune system (see, e.g.,Kucherlapati et al., U.S. Pat. No. 6,657,103).

In another preferred embodiment, the monoclonal antibody is a humanizedantibody. A humanized antibody is a chimeric antibody wherein the largemajority of the amino acid residues are derived from human antibodies,thus minimizing any potential immune reaction when delivered to a humansubject. In humanized antibodies, amino acid residues in thecomplementarity determining regions are replaced, at least in part, withresidues from a non-human species that confer a desired antigenspecificity or affinity. Such altered immunoglobulin molecules can bemade by any of several techniques known in the art, (e.g., Teng et al.,Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al.,Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92,3-16 (1982)), and are preferably made according to the teachings of PCTPublication WO92/06193 or EP 0239400, all of which are incorporatedherein by reference). Humanized antibodies can be commercially producedby, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex,Great Britain. For further reference, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992), all of which areincorporated herein by reference.

In another preferred embodiment, the monoclonal, chimeric, or humanizedantibodies described above may contain amino acid residues that do notnaturally occur in any antibody in any species in nature. These foreignresidues can be utilized, for example, to confer novel or modifiedspecificity, affinity or effector function on the monoclonal, chimericor humanized antibody. In another preferred embodiment, the antibodiesdescribed above may be conjugated to drugs for systemic pharmacotherapy,such as toxins, low-molecular-weight cytotoxic drugs, biologicalresponse modifiers, and radionuclides (see e.g., Kunz et al.,Calicheamicin derivative-carrier conjugates, US20040082764 A1).

In one embodiment, the antibody is an antibody that specifically bindsto the Aβ fragment of amyloid precursor protein or to other componentsof an amyloid plaque, and is useful in combating the accumulation ofamyloid plaques in the brain which characterize Alzheimer's disease.(See, e.g., U.S. Provisional Application 60/636,684.)

In another embodiment, antibodies of the present invention are directedagainst cell surface antigens expressed on target cells and/or tissuesin proliferative disorders such as cancer. In one embodiment, theantibody is an IgG1 anti-Lewis Y antibody. Lewis Y is a carbohydrateantigen with the structure Fuc

1→2Galβ1→4[Fuc

1→3]GlcNacβ1→3R (Abe et al. (1983) J. Biol. Chem., 258 11793-11797).Lewis Y antigen is expressed on the surface of 60% to 90% of humanepithelial tumors (including those of the breast, colon, lung, andprostate), at least 40% of which overexpress this antigen, and haslimited expression in normal tissues.

In order to target Ley and effectively target a tumor, an antibody withexclusive specificity to the antigen is ideally required. Thus,preferably, the anti-Lewis Y antibodies of the present invention do notcross-react with the type 1 structures (i.e., the lacto-series of bloodgroups (Lea and Leb)) and, preferably, do not bind other type 2 epitopes(i.e., neolacto-structure) like Lex and H-type 2 structures. An exampleof a preferred anti-Lewis Y antibody is designated hu3S193 (see U.S.Pat. Nos. 6,310,185; 6,518,415; 5,874,060, incorporated herein in theirentirety). The humanized antibody hu3S193 (Attia, M. A., et al.1787-1800) was generated by CDR-grafting from 3S193, which is a murinemonoclonal antibody raised against adenocarcinoma cell with exceptionalspecificity for Ley (Kitamura, K., 12957-12961). Hu3S193 not onlyretains the specificity of 3S193 for Ley but has also gained in thecapability to mediate complement dependent cytotoxicity (hereinafterreferred to as CDC) and antibody dependent cellular cytotoxicity(hereinafter referred to as ADCC) (Attia, M. A., et al. 1787-1800). Thisantibody targets Ley expressing xenografts in nude mice as demonstratedby biodistribution studies with hu3S193 labeled with 125I, 111In, or18F, as well as other radiolabels that require a chelating agent, suchas 111In, 99mTc, or 90Y (Clark, et al. 4804-4811).

In another embodiment, the antibody is one of the human anti-GDF-8antibodies termed Myo29, Myo28, and Myo22, and antibodies andantigen-binding fragments derived therefrom. These antibodies arecapable of binding mature GDF-8 with high affinity, inhibit GDF-8activity in vitro and in vivo as demonstrated, for example, byinhibition of ActRIIB binding and reporter gene assays, and may inhibitGDF-8 activity associated with negative regulation of skeletal musclemass and bone density. See, e.g., Veldman, et al, U.S. PatentApplication No. 20040142382.

Receptors

Another class of polypeptides that have been shown to be effective aspharmaceutical and/or commercial agents includes receptors. Receptorsare typically trans-membrane glycoproteins that function by recognizingan extra-cellular signaling ligand. Receptors typically have a proteinkinase domain in addition to the ligand recognizing domain, whichinitiates a signaling pathway by phosphorylating target intracellularmolecules upon binding the ligand, leading to developmental or metabolicchanges within the cell. In one embodiment, the receptors of interestare modified so as to remove the transmembrane and/or intracellulardomain(s), in place of which there may optionally be attached anIg-domain. In a preferred embodiment, receptors to be produced inaccordance with the present invention are receptor tyrosine kinases(RTKs). The RTK family includes receptors that are crucial for a varietyof functions numerous cell types (see, e.g., Yarden and Ullrich, Ann.Rev. Biochem. 57:433-478, 1988; Ullrich and Schlessinger, Cell61:243-254, 1990, incorporated herein by reference). Non-limitingexamples of RTKs include members of the fibroblast growth factor (FGF)receptor family, members of the epidermal growth factor receptor (EGF)family, platelet derived growth factor (PDGF) receptor, tyrosine kinasewith immunoglobulin and EGF homology domains-1 (TIE-1) and TIE-2receptors (Sato et al., Nature 376(6535):70-74 (1995), incorporatedherein be reference) and c-Met receptor, some of which have beensuggested to promote angiogenesis, directly or indirectly (Mustonen andAlitalo, J. Cell Biol. 129:895-898, 1995). Other non-limiting examplesof RTK's include fetal liver kinase 1 (FLK-1) (sometimes referred to askinase insert domain-containing receptor (KDR) (Terman et al., Oncogene6:1677-83, 1991) or vascular endothelial cell growth factor receptor 2(VEGFR-2)), fms-like tyrosine kinase-1 (Flt-1) (DeVries et al. Science255; 989-991, 1992; Shibuya et al., Oncogene 5:519-524, 1990), sometimesreferred to as vascular endothelial cell growth factor receptor 1(VEGFR-1), neuropilin-1, endoglin, endosialin, and Axl. Those ofordinary skill in the art will be aware of other receptors that canpreferably be expressed in accordance with the present invention.

In a particularly preferred embodiment, tumor necrosis factorinhibitors, in the form of tumor necrosis factor alpha and betareceptors (TNFR-1; EP 417,563 published Mar. 20, 1991; and TNFR-2, EP417,014 published Mar. 20, 1991) are expressed in accordance with thepresent invention (for review, see Naismith and Sprang, J Inflamm.47(1-2): 1-7 (1995-96), incorporated herein by reference). According toone embodiment, the tumor necrosis factor inhibitor comprises a solubleTNF receptor and preferably a TNFR-Ig. In one embodiment, the preferredTNF inhibitors of the present invention are soluble forms of TNFRI andTNFRII, as well as soluble TNF binding proteins, in another embodiment,the TNFR-Ig fusion is a TNFR:Fc, a term which as used herein refers to“etanercept,” which is a dimer of two molecules of the extracellularportion of the p75 TNF-.alpha. receptor, each molecule consisting of a235 amino acid Fc portion of human IgG.sub.1.

Growth Factors and Other Signaling Molecules

Another class of polypeptides that have been shown to be effective aspharmaceutical and/or commercial agents includes growth factors andother signaling molecules. Growth factors are typically glycoproteinsthat are secreted by cells and bind to and activate receptors on othercells, initiating a metabolic or developmental change in the receptorcell. In one embodiment, the protein of interest is an ActRIIB fusionpolypeptide comprising the extracellular domain of the ActRIIB receptiorand the Fc portion of an antibody (see, e.g., Wolfman, et al., ActRIIBfusion polypeptides and uses therefor, US2004/0223966 A1). In anotherembodiment, the growth factor may be a modified GDF-8 propeptide (see,e.g., Wolfman, et al., Modifed and stabilized GDF propeptides and usesthereof, US2003/0104406 A1). Alternatively, the protein of interestcould be a follistatin-domain-containing protein (see, e.g., Hill, etal., GASP1: a follistatin domain containing protein, US 2003/0162714 A1,Hill, et al., GASP1: a follistatin domain containing protein, US2005/0106154 A1, Hill, et al., Follistatin domain containing proteins,US 2003/0180306 A1).

Non-limiting examples of mammalian growth factors and other signalingmolecules include cytokines; epidermal growth factor (EGF);platelet-derived growth factor (PDGF); fibroblast growth factors (FGFs)such as aFGF and bFGF; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3,TGF-beta 4, or TGF-beta 5; insulin-like growth factor-I and -II (IGF-Iand IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (TLs), e.g., IL-1 to IL-10; tumornecrosis factor (TNF) alpha and beta; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor IX,tissue factor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA); bombesin; thrombin, hemopoietic growth factor;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-alpha);mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; neurotrophic factorssuch as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5,or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such asNGF-beta. One of ordinary skill in the art will be aware of other growthfactors or signaling molecules that can be expressed in accordance withthe present invention.

G-Protein Coupled Receptors

Another class of polypeptides that have been shown to be effective aspharmaceutical and/or commercial agents includes growth factors andother signaling molecules. G-protein coupled receptors (GPCRs) areproteins that have seven transmembrane domains. Upon binding of a ligandto a GPCR, a signal is transduced within the cell which results in achange in a biological or physiological property of the cell.

GPCRs, along with G-proteins and effectors (intracellular enzymes andchannels which are modulated by G-proteins), are the components of amodular signaling system that connects the state of intracellular secondmessengers to extracellular inputs. These genes and gene-products arepotential causative agents of disease.

Specific defects in the rhodopsin gene and the V2 vasopressin receptorgene have been shown to cause various forms of autosomal dominant andautosomal recessive retinitis pigmentosa, nephrogenic diabetesinsipidus. These receptors are of critical importance to both thecentral nervous system and peripheral physiological processes. The GPCRprotein superfamily now contains over 250 types of paralogues, receptorsthat represent variants generated by gene duplications (or otherprocesses), as opposed to orthologues, the same receptor from differentspecies. The superfamily can be broken down into five families: FamilyI, receptors typified by rhodopsin and the beta2-adrenergic receptor andcurrently represented by over 200 unique members; Family II, therecently characterized parathyroid hormone/calcitonin/secretin receptorfamily; Family III, the metabotropic glutamate receptor family inmammals; Family IV, the cAMP receptor family, important in thechemotaxis and development of D. discoideum; and Family V, the fungalmating pheromone receptors such as STE2.

GPCRs include receptors for biogenic amines, for lipid mediators ofinflammation, peptide hormones, and sensory signal mediators. The GPCRbecomes activated when the receptor binds its extracellular ligand.Conformational changes in the GPCR, which result from theligand-receptor interaction, affect the binding affinity of a G proteinto the GPCR intracellular domains. This enables GTP to bind withenhanced affinity to the G protein.

Activation of the G protein by GTP leads to the interaction of the Gprotein α subunit with adenylate cyclase or other second messengermolecule generators. This interaction regulates the activity ofadenylate cyclase and hence production of a second messenger molecule,cAMP. cAMP regulates phosphorylation and activation of otherintracellular proteins. Alternatively, cellular levels of other secondmessenger molecules, such as cGMP or eicosinoids, may be upregulated ordownregulated by the activity of GPCRs. The G protein a subunit isdeactivated by hydrolysis of the GTP by GTPase, and the α, β, and γsubunits reassociate. The heterotrimeric G protein then dissociates fromthe adenylate cyclase or other second messenger molecule generator.Activity of GPCR may also be regulated by phosphorylation of the intra-and extracellular domains or loops.

Glutamate receptors form a group of GPCRs that are important inneurotransmission. Glutamate is the major neurotransmitter in the CNSand is believed to have important roles in neuronal plasticity,cognition, memory, learning and some neurological disorders such asepilepsy, stroke, and neurodegeneration (Watson, S. and S. Arkinstall(1994) The G-Protein Linked Receptor Facts Book, Academic Press, SanDiego Calif., pp. 130-132). These effects of glutamate are mediated bytwo distinct classes of receptors termed ionotropic and metabotropic.Ionotropic receptors contain an intrinsic cation channel and mediatefast excitatory actions of glutamate. Metabotropic receptors aremodulatory, increasing the membrane excitability of neurons byinhibiting calcium dependent potassium conductances and both inhibitingand potentiating excitatory transmission of ionotropic receptors.Metabotropic receptors are classified into five subtypes based onagonist pharmacology and signal transduction pathways and are widelydistributed in brain tissues.

The vasoactive intestinal polypeptide (VIP) family is a group of relatedpolypeptides whose actions are also mediated by GPCRs. Key members ofthis family are VIP itself, secretin, and growth hormone releasingfactor (GRF). VIP has a wide profile of physiological actions includingrelaxation of smooth muscles, stimulation or inhibition of secretion invarious tissues, modulation of various immune cell activities. andvarious excitatory and inhibitory activities in the CNS. Secretinstimulates secretion of enzymes and ions in the pancreas and intestineand is also present in small amounts in the brain. GRF is an importantneuroendocrine agent regulating synthesis and release of growth hormonefrom the anterior pituitary (Watson, S. and S. Arkinstall supra, pp.278-283).

Following ligand binding to the GPCR, a conformational change istransmitted to the G protein, which causes the α-subunit to exchange abound GDP molecule for a GTP molecule and to dissociate from theβγ-subunits. The GTP-bound form of the α-subunit typically functions asan effector-modulating moiety, leading to the production of secondmessengers, such as cyclic AMP (e.g., by activation of adenylatecyclase), diacylglycerol or inositol phosphates. Greater than 20different types of α-subunits are known in man, which associate with asmaller pool of β and γ subunits. Examples of mammalian G proteinsinclude Gi, Go, Gq, Gs and Gt. G proteins are described extensively inLodish H. et al. Molecular Cell Biology, (Scientific American BooksInc., New York, N.Y., 1995), the contents of which is incorporatedherein by reference.

GPCRs are a major target for drug action and development. In fact,receptors have led to more than half of the currently known drugs(Drews, Nature Biotechnology, 1996, 14: 1516) and GPCRs represent themost important target for therapeutic intervention with 30% ofclinically prescribed drugs either antagonizing or agonizing a GPCR(Milligan, G. and Rees, S., (1999) TIPS, 20: 118-124). This demonstratesthat these receptors have an established, proven history as therapeutictargets.

In general, practitioners of the present invention will selected theirpolypeptide of interest, and will know its precise amino acid sequence.The techniques of the present invention have been successfully appliedto production of diverse polypeptides including, for example, a humanmonoclonal antibody directed to growth and differentiation factor 8(Examples 1, 3, 4, 7-14), humanized anti-Lewis Y antibody (Examples 5and 6), anti-ABeta (Example 15) and a dimeric Fc-fusion protein of tumornecrosis factor receptor (Example 16), indicating that the presentinvention will be useful for expression of a variety of differentpolypeptides and proteins. Any given protein that is to be expressed inaccordance with the present invention will have its own idiosyncraticcharacteristics and may influence the cell density or viability of thecultured cells, and may be expressed at lower levels than anotherpolypeptide or protein grown under identical culture conditions. One ofordinary skill in the art will be able to appropriately modify the stepsand compositions of the present invention in order to optimize cellgrowth and/or production of any given expressed polypeptide or protein.

Genetic Control Elements

As will be clear to those of ordinary skill in the art, genetic controlelements may be employed to regulate gene expression of the polypeptideor protein. Such genetic control elements should be selected to beactive in the relevant host cell. Control elements may be constitutivelyactive or may be inducible under defined circumstances. Induciblecontrol elements are particularly useful when the expressed protein istoxic or has otherwise deleterious effects on cell growth and/orviability. In such instances, regulating expression of the polypeptideor protein through inducible control elements may improve cellviability, cell density, and/or total yield of the expressed polypeptideor protein. A large number of control elements useful in the practice ofthe present invention are known and available in the art.

Representative constitutive mammalian promoters that may be used inaccordance with the present invention include, but are not limited to,the hypoxanthine phosphoribosyl transferase (HPTR) promoter, theadenosine deaminase promoter, the pyruvate kinase promoter, thebeta-actin promoter as well as other constitutive promoters known tothose of ordinary skill in the art. Additionally, viral promoters thathave been shown to drive constitutive expression of coding sequences ineukaryotic cells include, for example, simian virus promoters, herpessimplex virus promoters, papilloma virus promoters, adenoviruspromoters, human immunodeficiency virus (HIV) promoters, Rous sarcomavirus promoters, cytomegalovirus (CMV) promoters, the long terminalrepeats (LTRs) of Moloney murine leukemia virus and other retroviruses,the thymidine kinase promoter of herpes simplex virus as well as otherviral promoters known to those of ordinary skill in the art.

Inducible promoters drive expression of operably linked coding sequencesin the presence of an inducing agent and may also be used in accordancewith the present invention. For example, in mammalian cells, themetallothionein promoter is induces transcription of downstream codingsequences in the presence of certain metal ions. Other induciblepromoters will be recognized by and/or known to those of ordinary skillin the art.

In general, the gene expression sequence will also include 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Enhancerelements can optionally be used to increase expression levels of thepolypeptides or proteins to be expressed. Examples of enhancer elementsthat have been shown to function in mammalian cells include the SV40early gene enhancer, as described in Dijkema et al., EMBO J. (1985) 4:761 and the enhancer/promoter derived from the long terminal repeat(LTR) of the Rous Sarcoma Virus (RSV), as described in Gorman et al.,Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and human cytomegalovirus, asdescribed in Boshart et al., Cell (1985) 41:521.

Systems for linking control elements to coding sequences are well knownin the art (general molecular biological and recombinant DNA techniquesare described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989, which is incorporated herein byreference). Commercial vectors suitable for inserting preferred codingsequence for expression in various mammalian cells under a variety ofgrowth and induction conditions are also well known in the art.

Introduction of Coding Sequences and Related Control Elements into HostCells

Methods suitable for introducing into mammalian host cells nucleic acidssufficient to achieve expression of the polypeptides or proteins ofinterest are well known in the art. See, for example, Gething et al.,Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979);Levinson et al.; EP 117,060; and EP 117,058, all incorporated herein byreference.

For mammalian cells, preferred methods of transformation include thecalcium phosphate precipitation method of Graham and van der Erb,Virology, 52:456-457 (1978) or the lipofectamine™. (Gibco BRL) Method ofHawley-Nelson, Focus 15:73 (1193). General aspects of mammalian cellhost system transformations have been described by Axel in U.S. Pat. No.4,399,216 issued Aug. 16, 1983. For various techniques for transformingmammalian cells, see Keown et al., Methods in Enzymology (1989), Keownet al., Methods in Enzymology, 185:527-537 (1990), and Mansour et al.,Nature, 336:348-352 (1988). Non-limiting representative examples ofsuitable vectors for expression of polypeptides or proteins in mammaliancells include pcDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.5:1136-1142; pMClneo Poly-A, see Thomas, et al. (1987) Cell 51:503-512;and a baculovirus vector such as pAC 373 or pAC 610.

In preferred embodiments, the polypeptide or protein is stablytransfected into the host cell. However, one of ordinary skill in theart will recognize that the present invention can be used with eithertransiently or stably transfected mammalian cells.

Cells

Any mammalian cell or cell type susceptible to cell culture, and toexpression of polypeptides, may be utilized in accordance with thepresent invention. Non-limiting examples of mammalian cells that may beused in accordance with the present invention include BALB/c mousemyeloma line (NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6(CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells +/−DHFR (CHO, Urlaub and Chasin, Proc. Natl.Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1 587); humancervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al.,Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2). In a particularly preferred embodiment,the present invention is used in the culturing of and expression ofpolypeptides and proteins from CHO cell lines.

Additionally, any number of commercially and non-commercially availablehybridoma cell lines that express polypeptides or proteins may beutilized in accordance with the present invention. One skilled in theart will appreciate that hybridoma cell lines might have differentnutrition requirements and/or might require different culture conditionsfor optimal growth and polypeptide or protein expression, and will beable to modify conditions as needed.

As noted above, in many instances the cells will be selected orengineered to produce high levels of protein or polypeptide. Often,cells are genetically engineered to produce high levels of protein, forexample by introduction of a gene encoding the protein or polypeptide ofinterest and/or by introduction of control elements that regulateexpression of the gene (whether endogenous or introduced) encoding thepolypeptide of interest.

Certain polypeptides may have detrimental effects on cell growth, cellviability or some other characteristic of the cells that ultimatelylimits production of the polypeptide or protein of interest in some way.Even amongst a population of cells of one particular type engineered toexpress a specific polypeptide, variability within the cellularpopulation exists such that certain individual cells will grow betterand/or produce more polypeptide of interest. In certain preferredembodiments of the present invention, the cell line is empiricallyselected by the practitioner for robust growth under the particularconditions chosen for culturing the cells. In particularly preferredembodiments, individual cells engineered to express a particularpolypeptide are chosen for large-scale production based on cell growth,final cell density, percent cell viability, titer of the expressedpolypeptide or any combination of these or any other conditions deemedimportant by the practitioner.

Cell Culture Phase

Typical procedures for producing a polypeptide of interest include batchcultures and fed-batch cultures. Batch culture processes traditionallycomprise inoculating a large-scale production culture with a seedculture of a particular cell density, growing the cells under conditionsconducive to cell growth and viability, harvesting the culture when thecells reach a specified cell density, and purifying the expressedpolypeptide. Fed-batch culture procedures include an additional step orsteps of supplementing the batch culture with nutrients and othercomponents that are consumed during the growth of the cells. Apersistent and unsolved problem with traditional batch and fed-batchcultures is the production of metabolic waste products, which havedetrimental effects on cell growth, viability, and production ofexpressed polypeptides. Two metabolic waste products that haveparticularly detrimental effects are lactate and ammonium, which areproduced as a result of glucose and glutamine metabolism, respectively.In addition to the enzymatic production of ammonium as a result ofglutamine metabolism, ammonium also accumulates in cell cultures as aresult of non-metabolic degradation over time. The present inventionprovides an improved method of large-scale production of polypeptidesthat minimizes the detrimental effects of ammonium and lactate byslowing and even reversing the accumulation of these waste products incell cultures. One of ordinary skill in the art will recognize that thepresent invention can be employed in any system in which cells arecultured including, but not limited to, batch, fed-batch and perfusionsystems. In certain preferred embodiments of the present invention, thecells are grown in batch or fed-batch systems.

Media

Traditional media formulations, including commercially available mediasuch as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM],Sigma), have contained relatively high levels of glucose and glutaminein comparison to other amino acids. These components have been thoughtto be required in abundance since they are the primary metabolic energysources for the cells. However, rapid consumption of these nutrientsleads to the accumulation of lactate and ammonium as described above.Additionally, high initial levels of glucose and glutamine and thesubsequent accumulation of lactate and ammonium result in highosmolarity, a condition that by itself is often detrimental to cellgrowth, cell viability and the production of polypeptides.

The present invention provides a variety of media formulations that,when used in accordance with other culturing steps described herein,minimize and even reverse accumulation of lactate and ammonium. Mediaformulations of the present invention that have been shown to havebeneficial effects on cell growth and/or viability or on expression ofpolypeptide or protein include one or more of: i) a cumulative aminoacid amount per unit volume greater than about 70 mM, ii) a molarcumulative glutamine to cumulative asparagine ratio of less than about2, iii) a molar cumulative glutamine to cumulative total amino acidratio of less than about 0.2, iv) a molar cumulative inorganic ion tocumulative total amino acid ratio between about 0.4 to 1, and v) acombined cumulative amount of glutamine and asparagine per unit volumegreater than about 16 mM. One of ordinary skill in the art willunderstand that “cumulative”, as used above, refers to the total amountof a particular component or components added over the course of thecell culture, including components added at the beginning of the cultureand subsequently added components. One of ordinary skill in the art willunderstand that the media formulations of the present inventionencompass both defined and non-defined media.

Traditional media formulations begin with a relatively low level oftotal amino acids in comparison with the media formulations of thepresent invention. For example, the traditional cell culture mediumknown as DME-F12 (a 50:50 mixture of Dulbecco's Modified Eagle's mediumand Ham's F12 medium) has a total amino acid content of 7.29 mM, and thetraditional cell culture medium known as RPMI-1640 has a total aminoacid content of 6.44 mM (See e.g., H. J. Morton, In Vitro, 6:89-108(1970), R. G. Ham, Proc. Nat. Assoc. Sci. (USA), 53:288-293 (1965), G.E. Moore et al., J. Am. Medical Assn., 199:519-24 (1967), allincorporated herein by reference). In certain embodiments of the presentinvention, the amino acid concentration in the culture media ispreferably greater than about 70 mM. More preferably still, the mediaformulations of the present invention contain amino acid concentrationsgreater than about 70 mM in the starting media. It has been shown thatwhen amino acid concentrations of the starting media are in this range,cell density and titer are increased throughout the growth period of theculture (see Example 13).

Additionally, in certain embodiments of the present invention, the molarratio of glutamine to asparagine in the culture media is reducedcompared to other commercially and non-commercially available media.Preferably the molar ratio of glutamine to asparagine in the culturemedia is less than about two.

Additionally, in certain embodiments of the present invention, the molarratio of glutamine to total amino acids in the culture media is reducedcompared to other commercially and non-commercially available media.Preferably the molar ratio of glutamine to total amino acids in theculture media less than about 0.2.

An interesting and unexpected result of lowering the molar ratio ofglutamine to asparagine or to the total concentration of amino acids inthe starting media according to the present invention was that inaddition to an observed decrease in the accumulation of ammonium, adecrease in the accumulation of lactate was seen as well. In certainembodiments, the accumulated levels of ammonium and lactate are not onlylower than those in control cultures, but in fact actually decreaseafter an initial accumulation (for example, see Examples 3 and 7).

Boraston (U.S. Pat. No. 5,871,999) has disclosed a culture medium inwhich the molar ratio of total inorganic ions to total amino acids isbetween 1 and 10. Boraston showed that by providing culture medium inwhich the molar ratio of total inorganic ions to total amino acids is inthis range, aggregation of CHO cells grown in the medium is decreased.In another preferred embodiment of the present invention, the molarratio of total inorganic ions to total amino acids in the culture mediumis reduced even further, to between about 0.4 to 1. As shown in Example13, reducing this ratio from 1.75 to approximately 0.7 results in amarked increase in cell density and production of expressed polypeptideor protein throughout the growth period of the culture.

In another preferred embodiment of the present invention, the culturemedium contains a combined glutamine and asparagine concentration ofbetween about 16 and 36 mM. As shown in Example 14, Table 22, mediawhich contain a combined total concentration of glutamine and asparaginewithin this range exhibit higher titers of expressed polypeptide thanmedia which contain a combined total glutamine and asparagine outsidethis range. One of ordinary skill in the art will be able to choose theexact combined glutamine and asparagine concentration within this rangein order to optimize cell growth and/or viability and to maximize theproduction of the expressed polypeptide.

Furthermore, one of ordinary skill in the art will recognize that any ofthe conditions listed above may be used either singly or in variouscombinations with one another. By utilizing media formulation whichexhibit one, some or all of the above characteristics, one of ordinaryskill in the art will be able to optimize cell growth and/or viabilityand to maximize the production of the expressed polypeptide.

Any of these media formulations disclosed in the present invention mayoptionally be supplemented as necessary with hormones and/or othergrowth factors, particular ions (such as sodium, chloride, calcium,magnesium, and phosphate), buffers, vitamins, nucleosides ornucleotides, trace elements (inorganic compounds usually present at verylow final concentrations), amino acids, lipids, protein hydrolysates, orglucose or other energy source. In certain embodiments of the presentinvention, it may be beneficial to supplement the media with chemicalinductants such as hexamethylene-bis(acetamide) (“HMBA”) and sodiumbutyrate (“NaB”). These optional supplements may be added at thebeginning of the culture or may be added at a later point in order toreplenish depleted nutrients or for another reason. One of ordinaryskill in the art will be aware of any desirable or necessary supplementsthat may be included in the disclosed media formulations.

Providing a Mammalian Cell Culture

Various methods of preparing mammalian cells for production of proteinsor polypeptides by batch and fed-batch culture are well known in theart. As described above, a nucleic acid sufficient to achieve expression(typically a vector containing the gene encoding the polypeptide orprotein of interest and any operably linked genetic control elements)may be introduced into the host cell line by any number of well-knowntechniques. Typically, cells are screened to determine which of the hostcells have actually taken up the vector and express the polypeptide orprotein of interest. Traditional methods of detecting a particularpolypeptide or protein of interest expressed by mammalian cells includebut are not limited to immunohistochemistry, immunoprecipitation, flowcytometry, immunofluorescence microscopy, SDS-PAGE, Western blots,enzyme-linked immunosorbent assay (ELISA), high performance liquidchromatography (HPLC) techniques, biological activity assays andaffinity chromatography. One of ordinary skill in the art will be awareof other appropriate techniques for detecting expressed polypeptides orproteins. If multiple host cells express the polypeptide or protein ofinterest, some or all of the listed techniques can be used to determinewhich of the cells expresses that polypeptide or protein at the highestlevels.

Once a cell that expresses the polypeptide or protein of interest hasbeen identified, the cell is propagated in culture by any of the varietyof methods well-known to one of ordinary skill in the art. The cellexpressing the polypeptide or protein of interest is typicallypropagated by growing it at a temperature and in a medium that isconducive to the survival, growth and viability of the cell. The initialculture volume can be of any size, but is often smaller than the culturevolume of the production bioreactor used in the final production of thepolypeptide or protein of interest, and frequently cells are passagedseveral times in bioreactors of increasing volume prior to seeding theproduction bioreactor. The cell culture can be agitated or shaken toincrease oxygenation of the medium and dispersion of nutrients to thecells. Alternatively or additionally, special sparging devices that arewell known in the art can be used to increase and control oxygenation ofthe culture. In accordance with the present invention, one of ordinaryskill in the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor, including butnot limited to pH, temperature, oxygenation, etc.

The starting cell density in the production bioreactor can be chosen byone of ordinary skill in the art. In accordance with the presentinvention, the starting cell density in the production bioreactor can beas low as a single cell per culture volume. In preferred embodiments ofthe present invention, starting cell densities in the productionbioreactor can range from about 2×10² viable cells per mL to about2×10³, 2×10⁴, 2×10⁵, 2×10⁶, 5×10⁶ or 10×10⁶ viable cells per mL andhigher.

Initial and intermediate cell cultures may be grown to any desireddensity before seeding the next intermediate or final productionbioreactor. It is preferred that most of the cells remain alive prior toseeding, although total or near total viability is not required. In oneembodiment of the present invention, the cells may be removed from thesupernatant, for example, by low-speed centrifugation. It may also bedesirable to wash the removed cells with a medium before seeding thenext bioreactor to remove any unwanted metabolic waste products ormedium components. The medium may be the medium in which the cells werepreviously grown or it may be a different medium or a washing solutionselected by the practitioner of the present invention.

The cells may then be diluted to an appropriate density for seeding theproduction bioreactor. In a preferred embodiment of the presentinvention, the cells are diluted into the same medium that will be usedin the production bioreactor. Alternatively, the cells can be dilutedinto another medium or solution, depending on the needs and desires ofthe practitioner of the present invention or to accommodate particularrequirements of the cells themselves, for example, if they are to bestored for a short period of time prior to seeding the productionbioreactor.

Initial Growth Phase

Once the production bioreactor has been seeded as described above, thecell culture is maintained in the initial growth phase under conditionsconducive to the survival, growth and viability of the cell culture. Theprecise conditions will vary depending on the cell type, the organismfrom which the cell was derived, and the nature and character of theexpressed polypeptide or protein.

In accordance with the present invention, the production bioreactor canbe any volume that is appropriate for large-scale production ofpolypeptides or proteins. In a preferred embodiment, the volume of theproduction bioreactor is at least 500 liters. In other preferredembodiments, the volume of the production bioreactor is 1000, 2500,5000, 8000, 10,000, 12,000 liters or more, or any volume in between. Oneof ordinary skill in the art will be aware of and will be able to choosea suitable bioreactor for use in practicing the present invention. Theproduction bioreactor may be constructed of any material that isconducive to cell growth and viability that does not interfere withexpression or stability of the produced polypeptide or protein.

The temperature of the cell culture in the initial growth phase will beselected based primarily on the range of temperatures at which the cellculture remains viable. For example, during the initial growth phase,CHO cells grow well at 37° C. In general, most mammalian cells grow wellwithin a range of about 25° C. to 42° C. Preferably, mammalian cellsgrow well within the range of about 35° C. to 40° C. Those of ordinaryskill in the art will be able to select appropriate temperature ortemperatures in which to grow cells, depending on the needs of the cellsand the production requirements of the practitioner.

In one embodiment of the present invention, the temperature of theinitial growth phase is maintained at a single, constant temperature. Inanother embodiment, the temperature of the initial growth phase ismaintained within a range of temperatures. For example, the temperaturemay be steadily increased or decreased during the initial growth phase.Alternatively, the temperature may be increased or decreased by discreteamounts at various times during the initial growth phase. One ofordinary skill in the art will be able to determine whether a single ormultiple temperatures should be used, and whether the temperature shouldbe adjusted steadily or by discrete amounts.

The cells may be grown during the initial growth phase for a greater orlesser amount of time, depending on the needs of the practitioner andthe requirement of the cells themselves. In one embodiment, the cellsare grown for a period of time sufficient to achieve a viable celldensity that is a given percentage of the maximal viable cell densitythat the cells would eventually reach if allowed to grow undisturbed.For example, the cells may be grown for a period of time sufficient toachieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximalviable cell density.

In another embodiment the cells are allowed to grow for a defined periodof time. For example, depending on the starting concentration of thecell culture, the temperature at which the cells are grown, and theintrinsic growth rate of the cells, the cells may be grown for 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moredays. In some cases, the cells may be allowed to grow for a month ormore. The cells would be grown for 0 days in the production bioreactorif their growth in a seed bioreactor, at the initial growth phasetemperature, was sufficient that the viable cell density in theproduction bioreactor at the time of its inoculation is already at thedesired percentage of the maximal viable cell density. The practitionerof the present invention will be able to choose the duration of theinitial growth phase depending on polypeptide or protein productionrequirements and the needs of the cells themselves.

The cell culture may be agitated or shaken during the initial culturephase in order to increase oxygenation and dispersion of nutrients tothe cells. In accordance with the present invention, one of ordinaryskill in the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor during theinitial growth phase, including but not limited to pH, temperature,oxygenation, etc. For example, pH can be controlled by supplying anappropriate amount of acid or base and oxygenation can be controlledwith sparging devices that are well known in the art.

Shifting Culture Conditions

In accordance with the teaching of the present invention, at the end ofthe initial growth phase, at least one of the culture conditions may beshifted so that a second set of culture conditions is applied and ametabolic shift occurs in the culture. The accumulation of inhibitorymetabolites, most notably lactate and ammonia, inhibits growth. Ametabolic shift, accomplished by, e.g., a change in the temperature, pH,osmolality or chemical inductant level of the cell culture, may becharacterized by a reduction in the ratio of a specific lactateproduction rate to a specific glucose consumption rate. In onenon-limiting embodiment, the culture conditions are shifted by shiftingthe temperature of the culture. However, as is known in the art,shifting temperature is not the only mechanism through which anappropriate metabolic shift can be achieved. For example, such ametabolic shift can also be achieved by shifting other cultureconditions including, but not limited to, pH, osmolality, and sodiumbutyrate levels. As discussed above, the timing of the culture shiftwill be determined by the practitioner of the present invention, basedon polypeptide or protein production requirements or the needs of thecells themselves.

When shifting the temperature of the culture, the temperature shift maybe relatively gradual. For example, it may take several hours or days tocomplete the temperature change. Alternatively, the temperature shiftmay be relatively abrupt. For example, the temperature change may becomplete in less than several hours. Given the appropriate productionand control equipment, such as is standard in the commercial large-scaleproduction of polypeptides or proteins, the temperature change may evenbe complete within less than an hour.

The temperature of the cell culture in the subsequent growth phase willbe selected based primarily on the range of temperatures at which thecell culture remains viable and expresses recombinant polypeptides orproteins at commercially adequate levels. In general, most mammaliancells remain viable and express recombinant polypeptides or proteins atcommercially adequate levels within a range of about 25° C. to 42° C.Preferably, mammalian cells remain viable and express recombinantpolypeptides or proteins at commercially adequate levels within a rangeof about 25° C. to 35° C. Those of ordinary skill in the art will beable to select appropriate temperature or temperatures in which to growcells, depending on the needs of the cells and the productionrequirements of the practitioner.

In one embodiment of the present invention, the temperature of thesubsequent growth phase is maintained at a single, constant temperature.In another embodiment, the temperature of the subsequent growth phase ismaintained within a range of temperatures. For example, the temperaturemay be steadily increased or decreased during the subsequent growthphase. Alternatively, the temperature may be increased or decreased bydiscrete amounts at various times during the subsequent growth phase.One of ordinary skill in the art will understand that multiple discretetemperature shifts are encompassed in this embodiment. For example, thetemperature may be shifted once, the cells maintained at thistemperature or temperature range for a certain period of time, afterwhich the temperature may be shifted again—either to a higher or lowertemperature. The temperature of the culture after each discrete shiftmay be constant or may be maintained within a certain range oftemperatures.

In Example 16, data are shown that demonstrate the efficacy of employingtwo successive temperature changes, although it will be understood bythose of ordinary skill in the art that in accordance with the presentinvention, three or more successive temperature changes may be used toincrease cell viability or density and/or increase expression ofrecombinant polypeptides or proteins. The temperature or temperatureranges of the cell culture after each successive temperature shift maybe higher or lower than the temperature(s) or temperature range(s)preceding the shift. In a preferred embodiment of the present invention,each successive temperature or temperature range is lower than thepreceding temperature or temperature range.

Subsequent Production Phase

In accordance with the present invention, once the conditions of thecell culture have been shifted as discussed above, the cell culture ismaintained for a subsequent production phase under a second set ofculture conditions conducive to the survival and viability of the cellculture and appropriate for expression of the desired polypeptide orprotein at commercially adequate levels.

As discussed above, the culture may be shifted by shifting one or moreof a number of culture conditions including, but not limited to,temperature, pH, osmolality, and sodium butyrate levels. In oneembodiment, the temperature of the culture is shifted. According to thisembodiment, during the subsequent production phase, the culture ismaintained at a temperature or temperature range that is lower than thetemperature or temperature range of the initial growth phase. Forexample, during the subsequent production phase, CHO cells expressrecombinant polypeptides and proteins well within a range of 25° C. to35° C. As discussed above, multiple discrete temperature shifts may beemployed to increase cell density or viability or to increase expressionof the recombinant polypeptide or protein.

In accordance with the present invention, the cells may be maintained inthe subsequent production phase until a desired cell density orproduction titer is reached. In one embodiment, the cells are maintainedin the subsequent production phase until the titer to the recombinantpolypeptide or protein reaches a maximum. In other embodiments, theculture may be harvested prior to this point, depending on theproduction requirement of the practitioner or the needs of the cellsthemselves. For example, the cells may be maintained for a period oftime sufficient to achieve a viable cell density of 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percentof maximal viable cell density. In some cases, it may be desirable toallow the viable cell density to reach a maximum, and then allow theviable cell density to decline to some level before harvesting theculture. In an extreme example, it may be desirable to allow the viablecell density to approach or reach zero before harvesting the culture.

In another embodiment of the present invention, the cells are allowed togrow for a defined period of time during the subsequent productionphase. For example, depending on the concentration of the cell cultureat the start of the subsequent growth phase, the temperature at whichthe cells are grown, and the intrinsic growth rate of the cells, thecells may be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more days. In some cases, the cells may beallowed to grow for a month or more. The practitioner of the presentinvention will be able to choose the duration of the subsequentproduction phase depending on polypeptide or protein productionrequirements and the needs of the cells themselves.

In certain cases, it may be beneficial or necessary to supplement thecell culture during the subsequent production phase with nutrients orother medium components that have been depleted or metabolized by thecells. For example, it might be advantageous to supplement the cellculture with nutrients or other medium components observed to have beendepleted during monitoring of the cell culture (see ‘Monitoring CultureConditions’ section below). Alternatively or additionally, it may bebeneficial or necessary to supplement the cell culture prior to thesubsequent production phase. As non-limiting examples, it may bebeneficial or necessary to supplement the cell culture with hormonesand/or other growth factors, particular ions (such as sodium, chloride,calcium, magnesium, and phosphate), buffers, vitamins, nucleosides ornucleotides, trace elements (inorganic compounds usually present at verylow final concentrations), amino acids, lipids, or glucose or otherenergy source.

These supplementary components may all be added to the cell culture atone time, or they may be provided to the cell culture in a series ofadditions. In one embodiment of the present invention, the supplementarycomponents are provided to the cell culture at multiple times inproportional amounts. In another embodiment, it may be desirable toprovide only certain of the supplementary components initially, andprovide the remaining components at a later time. In yet anotherembodiment of the present invention, the cell culture is fed continuallywith these supplementary components.

In accordance with the present invention, the total volume added to thecell culture should optimally be kept to a minimal amount. For example,the total volume of the medium or solution containing the supplementarycomponents added to the cell culture may be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume of the cell cultureprior to providing the supplementary components.

The cell culture may be agitated or shaken during the subsequentproduction phase in order to increase oxygenation and dispersion ofnutrients to the cells. In accordance with the present invention, one ofordinary skill in the art will understand that it can be beneficial tocontrol or regulate certain internal conditions of the bioreactor duringthe subsequent growth phase, including but not limited to pH,temperature, oxygenation, etc. For example, pH can be controlled bysupplying an appropriate amount of acid or base and oxygenation can becontrolled with sparging devices that are well known in the art.

Monitoring Culture Conditions

In certain embodiments of the present invention, the practitioner mayfind it beneficial or necessary to periodically monitor particularconditions of the growing cell culture. Monitoring cell cultureconditions allows the practitioner to determine whether the cell cultureis producing recombinant polypeptide or protein at suboptimal levels orwhether the culture is about to enter into a suboptimal productionphase. In order to monitor certain cell culture conditions, it will benecessary to remove small aliquots of the culture for analysis. One ofordinary skill in the art will understand that such removal maypotentially introduce contamination into the cell culture, and will takeappropriate care to minimize the risk of such contamination.

As non-limiting example, it may be beneficial or necessary to monitortemperature, pH, cell density, cell viability, integrated viable celldensity, lactate levels, ammonium levels, osmolarity, or titer of theexpressed polypeptide or protein. Numerous techniques are well known inthe art that will allow one of ordinary skill in the art to measurethese conditions. For example, cell density may be measured using ahemacytometer, a Coulter counter, or Cell density examination (CEDEX).Viable cell density may be determined by staining a culture sample withTrypan blue. Since only dead cells take up the Trypan blue, viable celldensity can be determined by counting the total number of cells,dividing the number of cells that take up the dye by the total number ofcells, and taking the reciprocal. HPLC can be used to determine thelevels of lactate, ammonium or the expressed polypeptide or protein.Alternatively, the level of the expressed polypeptide or protein can bedetermined by standard molecular biology techniques such as coomassiestaining of SDS-PAGE gels, Western blotting, Bradford assays, Lowryassays, Biuret assays, and UV absorbance. It may also be beneficial ornecessary to monitor the post-translational modifications of theexpressed polypeptide or protein, including phosphorylation andglycosylation.

Isolation of Expressed Polypeptide

In general, it will typically be desirable to isolate and/or purifyproteins or polypeptides expressed according to the present invention.In a preferred embodiment, the expressed polypeptide or protein issecreted into the medium and thus cells and other solids may be removed,as by centrifugation or filtering for example, as a first step in thepurification process. This embodiment is particularly useful when usedin accordance with the present invention, since the methods andcompositions described herein result in increased cell viability. As aresult, fewer cells die during the culture process, and fewerproteolytic enzymes are released into the medium which can potentiallydecrease the yield of the expressed polypeptide or protein.

Alternatively, the expressed polypeptide or protein is bound to thesurface of the host cell. In this embodiment, the media is removed andthe host cells expressing the polypeptide or protein are lysed as afirst step in the purification process. Lysis of mammalian host cellscan be achieved by any number of means well known to those of ordinaryskill in the art, including physical disruption by glass beads andexposure to high pH conditions.

The polypeptide or protein may be isolated and purified by standardmethods including, but not limited to, chromatography (e.g., ionexchange, affinity, size exclusion, and hydroxyapatite chromatography),gel filtration, centrifugation, or differential solubility, ethanolprecipitation or by any other available technique for the purificationof proteins (See, e.g., Scopes, Protein Purification Principles andPractice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S. J.and Hames, B. D. (eds.), Protein Expression: A Practical Approach,Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J.N. (eds.), Guide to Protein Purification: Methods in Enzymology (Methodsin Enzymology Series, Vol 182), Academic Press, 1997, all incorporatedherein by reference). For immunoaffinity chromatography in particular,the protein may be isolated by binding it to an affinity columncomprising antibodies that were raised against that protein and wereaffixed to a stationary support. Alternatively, affinity tags such as aninfluenza coat sequence, poly-histidine, or glutathione-S-transferasecan be attached to the protein by standard recombinant techniques toallow for easy purification by passage over the appropriate affinitycolumn. Protease inhibitors such as phenyl methyl sulfonyl fluoride(PMSF), leupeptin, pepstatin or aprotinin may be added at any or allstages in order to reduce or eliminate degradation of the polypeptide orprotein during the purification process. Protease inhibitors areparticularly desired when cells must be lysed in order to isolate andpurify the expressed polypeptide or protein. One of ordinary skill inthe art will appreciate that the exact purification technique will varydepending on the character of the polypeptide or protein to be purified,the character of the cells from which the polypeptide or protein isexpressed, and the composition of the medium in which the cells weregrown.

Pharmaceutical Formulations

In certain preferred embodiments of the invention, produced polypeptidesor proteins will have pharmacologic activity and will be useful in thepreparation of pharmaceuticals. Inventive compositions as describedabove may be administered to a subject or may first be formulated fordelivery by any available route including, but not limited to parenteral(e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial,opthalmic, transdermal (topical), transmucosal, rectal, and vaginalroutes. Inventive pharmaceutical compositions typically include apurified polypeptide or protein expressed from a mammalian cell line, adelivery agent (i.e., a cationic polymer, peptide molecular transporter,surfactant, etc., as described above) in combination with apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS). In all cases, the composition should be sterile and should befluid to the extent that easy syringability exists. Preferredpharmaceutical formulations are stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. In general, therelevant carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thepurified polypeptide or protein in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the purified polypeptide or protein expressedfrom a mammalian cell line into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, thepurified polypeptide or protein can be incorporated with excipients andused in the form of tablets, troches, or capsules, e.g., gelatincapsules. Oral compositions can also be prepared using a fluid carrierfor use as a mouthwash. Pharmaceutically compatible binding agents,and/or adjuvant materials can be included as part of the composition.The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the inventive compositions comprisinga purified polypeptide or protein expressed from a mammalian cell lineand a delivery agent are preferably delivered in the form of an aerosolspray from a pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer. Thepresent invention particularly contemplates delivery of the compositionsusing a nasal spray, inhaler, or other direct delivery to the upperand/or lower airway. Intranasal administration of DNA vaccines directedagainst influenza viruses has been shown to induce CD8 T cell responses,indicating that at least some cells in the respiratory tract can take upDNA when delivered by this route, and the delivery agents of theinvention will enhance cellular uptake. According to certain embodimentsof the invention the compositions comprising a purified polypeptideexpressed from a mammalian cell line and a delivery agent are formulatedas large porous particles for aerosol administration.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the purified polypeptide or protein anddelivery agents are formulated into ointments, salves, gels, or creamsas generally known in the art.

The compositions can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

In one embodiment, the compositions are prepared with carriers that willprotect the polypeptide or protein against rapid elimination from thebody, such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active polypeptide or proteincalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier.

The polypeptide or protein expressed according to the present inventioncan be administered at various intervals and over different periods oftime as required, e.g., one time per week for between about 1 to 10weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or6 weeks, etc. The skilled artisan will appreciate that certain factorscan influence the dosage and timing required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Generally, treatment of a subjectwith a polypeptide or protein as described herein can include a singletreatment or, in many cases, can include a series of treatments. It isfurthermore understood that appropriate doses may depend upon thepotency of the polypeptide or protein and may optionally be tailored tothe particular recipient, for example, through administration ofincreasing doses until a preselected desired response is achieved. It isunderstood that the specific dose level for any particular animalsubject may depend upon a variety of factors including the activity ofthe specific polypeptide or protein employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

The present invention includes the use of inventive compositions fortreatment of nonhuman animals. Accordingly, doses and methods ofadministration may be selected in accordance with known principles ofveterinary pharmacology and medicine. Guidance may be found, forexample, in Adams, R. (ed.), Veterinary Pharmacology and Therapeutics,8^(th) edition, Iowa State University Press; ISBN: 0813817439; 2001.

Inventive pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

The foregoing description is to be understood as being representativeonly and is not intended to be limiting. Alternative methods andmaterials for implementing the invention and also additionalapplications will be apparent to one of skill in the art, and areintended to be included within the accompanying claims.

EXAMPLES Example 1 Enhanced Medium 1 for Anti-GDF-8 Fed-Batch Process

Traditional fed-batch processes for cultivating cell lines have severaldrawbacks including the time and effort required to administer the feedsand the need for special equipment in large-scale bioreactors. Theobjective was to develop a batch media for the production of proteins ofinterest in large-scale bioreactors that requires minimal feeds.

Materials and Methods

STRAINS AND MEDIA: Chinese Hamster Ovary (“CHO”) cells were engineeredto express a monoclonal antibody against growth and differentiationfactor 8 (“anti-GDF-8 cells”) (see Veldman et al., NeutralizingAntibodies Against GDF-8 and Uses Therefor, US20040142382 A1).Anti-GDF-8 cells were used to test a new batch media. Medium 1 andMedium 2 were compared for their abilities to support high cell densityand viability. The compositions of these media, as well as Medium 3 arelisted in Table 1. Media are made by adding all the components save forFeSO₄.7H₂O. The media is then adjusted to pH 7.25, the osmolarity isrecorded and FeSO₄.7H₂O are then added.

CULTURE CONDITIONS: For flask experiments, anti-GDF-8 cells were grownin shake flasks and passaged three times. For bioreactor experiments,anti-GDF-8 cells were grown in media for 12 days, supplemented dailywith either 2% by volume of 20× Medium 4 feed medium (Table 3) or 3% byvolume of 16× Medium 4 (Table 4) after day 5. For the first 4 days,cells were grown at 37° C. On day 5, cells were shifted to 31° C.

SAMPLE ANALYSIS: Daily samples were taken from the cultures and wereanalyzed for amino acid, vitamin, iron, phosphate, glucose and glutaminelevels. TABLE 1 Compositions of Medium 1, Medium 2 and Medium 3. Medium1 Medium 2 Medium 3 Amino Acids mg/L mM mg/L mM mg/L mM alanine 96.031.08 17.80 0.20 24.87 0.28 arginine 1186.99 6.82 347.97 2.00 423.43 2.43asparagine.H₂O 713.59 4.76 75.00 0.50 173.90 1.16 aspartic acid 318.532.39 26.20 0.20 52.72 0.40 cysteine.HCl.H₂O 70.01 0.40 70.19 0.40 70.010.40 cystine.2HCl 297.09 0.95 62.25 0.20 62.09 0.20 glutamic acid 158.591.08 29.40 0.20 41.08 0.28 glutamine 1892.40 12.96 1163.95 7.97 1162.407.96 glycine 95.88 1.28 30.00 0.40 35.92 0.48 histidine.HCl.H₂O 369.101.76 46.00 0.22 75.27 0.36 isoleucine 623.63 4.76 104.99 0.80 151.901.16 leucine 852.31 6.51 104.99 0.80 172.69 1.32 lysine.HCl 945.96 5.20145.99 0.80 218.38 1.20 methionine 291.82 1.96 29.80 0.20 53.55 0.36phenylalanine 428.62 2.60 65.99 0.40 98.81 0.60 proline 372.25 3.2468.99 0.60 96.40 0.84 serine 904.71 8.62 126.00 1.20 273.07 2.60threonine 513.39 4.31 94.99 0.80 132.81 1.12 tryptophan 159.32 0.7816.00 0.08 28.99 0.14 tyrosine.2Na.2H₂O 560.81 2.15 103.79 0.40 145.100.56 valine 505.36 4.32 93.99 0.80 131.17 1.12 Vitamins mg/L μM mg/L μMmg/L μM biotin 2.00 8.21 0.20 0.82 0.36 1.49 calcium pantothenate 22.0246.27 2.24 4.71 4.03 8.47 choline chloride 87.67 630.74 8.98 64.60 16.11115.92 folic acid 25.95 58.84 2.65 6.01 4.76 10.80 inositol 123.39685.47 12.60 69.99 22.64 125.79 nicotinamide 19.60 160.70 2.02 16.563.61 29.62 pyridoxal.HCl 1.99 9.83 2.00 9.85 1.99 9.83 pyridoxine.HCl18.06 87.67 0.03 0.15 1.67 8.10 riboflavin 2.20 5.85 0.22 0.59 0.40 1.06thiamine.HCl 21.51 63.84 2.17 6.44 3.92 11.64 vitamin B12 6.93 5.12 0.780.58 1.34 0.99 Inorganic Salts mg/L mM mg/L mM mg/L mM CaCl₂ 115.78 1.04116.09 1.05 115.78 1.04 KCl 310.94 4.17 311.77 4.18 310.94 4.17 Na₂HPO₄70.81 0.50 70.99 0.50 70.81 0.50 NaCl 1104.96 18.92 5539.00 94.853704.96 63.44 NaH₂PO₄.H₂O 636.33 4.61 62.49 0.45 114.33 0.83 MgSO₄ 48.700.41 48.83 0.41 48.70 0.41 MgSO₄.7H₂O 0.03 95.00 8.60 95.00 MgCl₂ 28.530.30 28.61 0.30 28.53 0.30 NaHCO₃ 2000.00 23.81 2440.00 29.04 2440.0029.04 Trace Elements μg/L nM μg/L nM μg/L nM Sodium Selenite 28.00161.94 5.00 28.92 7.00 40.49 Fe(NO₃)₃.9H₂O 49.86 123.42 50.00 123.7549.86 123.42 CuSO₄ 2.69 16.80 0.80 5.00 0.97 6.06 CuSO₄.5H₂O 11.24 45.007.49 30.00 FeSO₄.7H₂O 2503.85 9006.64 839.96 3021.45 1542.85 5549.81ZnSO₄.7H₂O 2734.77 9528.82 429.96 1498.12 1383.80 4821.59 MnSO₄.H₂O 0.261.51 0.17 1.01 Na2SiO3.9H₂O 210.00 739.27 140.00 492.84(NH4)₆Mo₇O₂₄.4H₂O 1.86 1.50 1.24 1.00 NH₄VO₃ 0.98 8.33 0.65 5.56NiSO₄.6H₂O 0.20 0.74 0.13 0.49 SnCl₂.2H₂O 0.18 0.80 0.12 0.53 OtherComponents mg/L μM mg/L μM mg/L μM Hydrocortisone 0.23 0.64 0.04 0.100.09 0.24 Putrescine.2HCl 6.48 40.22 1.08 6.70 2.48 15.39 linoleic acid0.22 0.80 0.04 0.14 0.06 0.20 thioctic acid 0.56 2.73 0.10 0.49 0.140.69 D-glucose (Dextrose) 16039.43 89107.92 6150.72 34170.64 11042.2461345.76 PVA 2560.00 2400.00 2520.00 Insulin 54.00 10.00 14.00 SodiumPyruvate 54.85 498.63 55.00 499.95 54.85 498.63Results and Conclusions

FIG. 1 shows that growth rate of anti-GDF-8 cells was similar in bothMedium 1 and Medium 2 in the flask experiments.

FIG. 2 shows that in bioreactors, Medium 1 exhibited a significantincrease in final cell density and viability over Medium 3. The finaltiter also increased significantly, from 551 mg/L for the platformprocess to 976 mg/L with Medium 1 (data not shown). Temperature wasshifted from 37° C. to 31° C. on day 5. Due to the unexpected high cellgrowth, the cultures were fed daily after day 5 with either 2% by volumeof 20× Medium 4 or 3% by volume of 16× Medium 4. Thus, this is not atrue batch experiment as originally intended. Asparagine and thiaminewere supplemented in the feed media beginning on day 10.

In developing a concentrated batch media, several possible concerns needto be considered. First, concentrated nutrients might prove toxic to thecells. In the media developed in this Example, all, nutrients andcomponents were determined to be below the toxicity limits (data notshown).

Second, the concentrated batch media necessarily has a higher osmolaritythan non-concentrated media, which has been shown to have detrimentaleffects on cell growth and viability. This problem can be circumventedby lowering the amount of NaCl in the starting media. Furthermore, theconcentrated batch media contains insufficient levels of glucose tosustain growth for the entire culture period. Thus, cultures weresupplemented daily after day 5 with a glucose feed.

Third, insulin and glutamine are susceptible to degradation during the12 day culture period. Thus, the culture was supplemented with thesecomponents in addition to glucose.

Finally, iron will precipitate out of solution containing highconcentrations of phosphate at high pH. This problem can be circumventedby adding iron at the end of the media preparation process, after the pHhas been adjusted to an appropriate level.

Example 2 Development of Concentrated Feed Medium (Medium 5) forAnti-GDF-8 Cells in Fed-Batch Process

In Example 1, a batch process for culturing anti-GDF-8 cells usingMedium 1 was developed. Due to the high cell density that resultedduring the process, it was determined that supplementation of nutrientsin addition to glucose and glutamine was still advantageous. However,supplementing the batch with 8× Medium 4 feed media would result inexcessive dilution of the culture. A more concentrated feed media wasdeveloped in order to circumvent this problem.

Materials and Methods and Results

Table 2 lists the compositions of Medium 4A-1, Medium 4B, Trace B andTrace D used in the formulations of Tables 3-7. TABLE 2 Compositions ofMedium 4A-1, Medium 4B, Trace B and Trace D used in the formulations ofTables 3-7. Medium 4A-1 Medium 4B Trace Elements B Trace Elements DAmino Acids mg/L mM mg/L mM mg/L mM mg/L mM alanine 17.80 0.20 arginine191.00 1.10 asparagine.H₂O 135.00 0.90 aspartic acid 66.50 0.50 glutamicacid 29.40 0.20 glycine 15.00 0.20 histidine.HCl.H₂O 73.50 0.35isoleucine 118.00 0.90 leucine 170.00 1.30 lysine.HCl 182.00 1.00methionine 59.60 0.40 phenylalanine 82.50 0.50 proline 69.00 0.60 serine158.00 1.50 threonine 95.20 0.80 tryptophan 32.60 0.16 tyrosine.2Na.2H₂O104.00 0.40 valine 93.60 0.80 Vitamins mg/L μM mg/L μM mg/L mM mg/L mMbiotin 0.41 1.68 calcium pantothenate 4.50 9.45 choline chloride 17.90128.78 folic acid 5.30 12.02 inositol 25.20 140.00 nicotinamide 4.0032.79 pyridoxine.HCl 4.10 19.90 riboflavin 0.45 1.20 thiamine.HCl 4.4013.06 vitamin B12 1.40 1.03 Trace Elements μg/L nM mg/L μM μg/L nM μg/LnM (NH₄)₆Mo₇O₂₄.4H₂O 1.24 1.00 CuSO₄ 0.43 2.69 CuSO₄.5H₂O 7.49 30.00FeSO₄.7H₂O 834 3000 MnSO₄.H₂O 0.17 1.01 Na₂SiO₃.9H₂O 140.00 492.84NH₄VO₃ 0.65 5.56 NiSO₄.6H₂O 0.13 0.49 SnCl₂.2H₂O 0.12 0.53 ZnSO₄.7H₂O230.00 801.39 863 3007 Other Components μg/L nM μg/L nM μg/L nM μg/L nMlinoleic acid 42.00 0.15 thioctic acid 105.00 0.51 D-glucose (Dextrose)1000000 5555.5620× Medium 4.

The first concentrated media was developed as 20× Medium 4. The mediaformulation for 20× Medium 4 is provided in Table 3. TABLE 3 20X Medium4 feed media worksheet. Part Component Amount Unit I Medium 4A-1 31.120g/L Nucellin ™ 40.000 ml/L H/P stock 20.000 ml/L Selenite Stock 2.000ml/L PVA 2.400 g/L NaH₂PO₄.H₂O 2.610 g/L MgSO₄.7H₂O 0.430 g/L Asparticacid 1.330 g/L Glutamic acid 0.588 g/L Linoleic acid 0.840 ml/L Thiocticacid 2.100 ml/L Tyrosine.2Na (Mw 225) 1.790 g/L 1000X Trace B 6.000 ml/LGlucose 100.000 g/L Glutamine 14.600 g/L pH to 7.0 Record Osmolarity1064.000 mOsm II Cysteine (400 mM) Add 108 ml Trace D, 0.25 g FeSO₄.7H₂Oto 280 ml Cysteine Stock III Folic acid 720 ml 6 mM Folic acidNote:Nucellin ™ is manufactured by Eli Lilly (Indianapolis, IN, USA);H/P stock = 0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine.2HCl.

The media formulation consists of 3 parts: I, II, III. Part I is theconcentrated version of 8× Medium 4 with the individual components ofMedium 4B except folic acid due to the concerns of the solubility ofthis vitamin. Part II is iron stock, Trace D and acidic cysteine, toavoid possible precipitation of iron if added in part I. Part III isfolic acid stock. Part I is added 2% by volume daily starting on day 5and parts II and III are added once on day 5 together with Part I.

The final pH of the feed media was adjusted to 7.0 and osmolarity wasabout 1064 mOsm. A 2% feed will result in a 2 g/L glucose, a 2 mMGlutamine and a 14 mOsm osmolarity increase to the culture.

2. 16× Medium 4.

To reduce the increase in osmolarity, the feed media was changed from20× Medium 4 (2% by volume daily) to 16× Medium 4 (3% by volume daily).The media formulation for 16× Medium 4 is provided in Table 4. TABLE 416X Medium 4 feed media worksheet. Part Component Amount Unit I Medium4A-1 24.896 g/L Nucellin ™ 32.000 ml/L H/P stock 16.000 ml/L SeleniteStock 1.600 ml/L PVA 2.400 g/L NaH₂PO₄.H₂O 2.088 g/L MgSO₄.7H₂O 0.344g/L Aspartic acid 1.064 g/L Glutamic acid 0.470 g/L Linoleic acid 0.672ml/L Thioctic acid 1.680 ml/L Tyrosine.2Na (Mw 225) 1.432 g/L 1000XTrace B 9.000 ml/L Glutamine 6.280 g/L pH to 7.0 Record Osmolarity295.000 mOsm II Cysteine (400 mM) Add 108 ml Trace D, 0.25 g FeSO₄.7H₂Oto 280 ml Cysteine Stock III Folic acid 720 ml 6 mM Folic acidNote:Nucellin ™ is manufactured by Eli Lilly (Indianapolis, IN, USA);H/P stock = 0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine.2HCl.

In this modified 16× Medium 4, glucose was also eliminated to furtherreduce the osmolarity and give some flexibility of the glucose feed.Total osmolarity of the feed media is now 295 mOsm.

3. 16× Medium 4.

Changes were made to the 16× Medium 4 formulation. Iron stock solutionwas added in the feed resulting in a 0.45 μM addition each feed.Additionally, glucose was added back to give a 1.5 g/L addition everyfeed. The media formulation for this modified 16× Medium 4 is providedin Table 5. TABLE 5 16X Medium 4 feed media worksheet. Part ComponentAmount Unit I Medium 4A-1 24.896 g/L Nucellin ™ 32.000 ml/L H/P stock16.000 ml/L Selenite Stock 1.600 ml/L PVA 2.400 g/L NaH₂PO₄.H₂O 2.088g/L MgSO₄.7H₂O 0.344 g/L Aspartic acid 1.064 g/L Glutamic acid 0.470 g/LLinoleic acid 0.672 ml/L Thioctic acid 1.680 ml/L Tyrosine.2Na (Mw 225)1.432 g/L 1000X Trace B 9.000 ml/L Glucose 50.000 g/L Glutamine 7.300g/L pH to 7.0 FeSO₄.7H₂O (1 mM stock) 15.000 ml/L Record Osmolarity607.000 mOsm II Folic acid 720 ml 6 mM Folic acid III Cysteine (400 mM)Add 108 ml Trace D, 0.25 g FeSO₄.7H₂O tol 280 m Cysteine StockNote:Nucellin ™ is manufactured by Eli Lilly (Indianapolis, IN, USA);H/P stock = 0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine.2HCl.4. 16× Medium 4.

Here, the feed media (16× Medium 4) was made in combined media insteadof 3 separate feeds as in the last several batches. Tests were done toensure that folic acid could be dissolved at the concentration requiredand that neither iron nor folic acid precipitated out of solution afterstorage at either 4° C. or at room temperature for 6 days. The mediaformulation for the combined 16× Medium 4 is provided in Table 6. TABLE6 16X Medium 4 feed media worksheet. Component Amount Unit Medium 4A-124.896 g/L Nucellin ™ 32.000 ml/L H/P stock 16.000 ml/L Selenite Stock1.600 ml/L PVA 2.400 g/L NaH₂PO₄.H₂O 2.088 g/L MgSO₄.7H₂O 0.344 g/LAspartic acid 1.064 g/L Glutamic acid 0.470 g/L Linoleic acid 0.672 ml/LThioctic acid 1.680 ml/L Tyrosine.2Na (Mw 225) 1.432 g/L Glucose 66.700g/L Glutamine 7.300 g/L Folic acid 70.560 mg/L Acidic cysteine (400 mM)6.250 ml/L FeSO₄ Stock (1 mM) 23.000 ml/L 1000x Trace B 9.000 ml/L 1000xTrace D 3.300 ml/L pH expected 6.11 Adjust to 7.0 Record Osmolarity724.000 mOsmNote:Nucellin ™ is manufactured by Eli Lilly (Indianapolis, IN, USA);H/P stock = 0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine.2HCl.

The final osmolarity of the media is 724 mOsm, with a daily glucoseaddition of 2 g/L and glutamine addition of 1.5 mM.

5. 12× Medium 4.

Here, several changes were made to the feed media. Medium 4B powder wasused instead of addition of each individual ingredient in Medium 4B.Medium 4B powder was mixed with glucose and dissolved separately underbasic conditions by titrating the solution to pH 10.25. Additionalasparagine and thiamine were added since the amino acid and vitaminanalysis results showed these two components were exhausted by the endof fed-batch process. Use of 12× Medium 4 further reduced the osmolarityincrease when fed to the culture. The media formulation for 12× Medium 4is provided in Table 7. TABLE 7 12X Medium 4 feed media worksheet.Component Amount Unit Medium 4A-1 18.672 g/L Nucellin ™ 24.000 ml/L H/Pstock 12.000 ml/L Selenite Stock 1.200 ml/L PVA 2.400 g/L Asparagine.H2O1.620 g/L NaH2PO4.H2O 1.566 g/L MgSO4.7H2O 0.258 g/L Glutamine 5.475 g/LThiamine 0.040 g/L Predissolved Medium 4B & ˜175 ml/L Glucose Acidiccysteine(400 mM) 4.688 ml/L Record pH Adjust pH to 7.2 with 5N HCl FeSO₄Stock (1 mM) 17.250 ml/L 1000x Trace B 6.750 ml/L 1000x Trace D 2.475ml/L Record pH (expect 7.18) Record Osm 566.000 Predissolved Medium 4B &Glucose * (for 1 L feed media) Water 150 ml Mix Medium 4B (14.5 g) Addin with glucose (38.3 g) Adjust pH using 25% NaOH until dissolved (pHabout 10.25)Note:Nucellin ™ is manufactured by Eli Lilly (Indianapolis, IN, USA); H/Pstock = 0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine.2HCl.

The final osmolarity is 566 mOsm. A daily feed of 4% by volume gives anapproximate osmolarity increase of 8.6, an increase in glucose of 2 g/Land an increase in glutamine of 1.5 mM. The 12× Medium 4 mediaformulation is also known as Medium 5. Medium 5 is easy to make comparedto 20× Medium 4 or 16× Medium 4, and stable over 10 days either at roomtemperature or at 4° C. (data not shown).

Example 3 Glutamine Starvation Fed-Batch Process for Anti-GDF-8 CellCulture

CHO cells require glutamine in the starting media to survive.Traditionally, initial glutamine levels are high and glutamine is feddaily after day 5 until the end of the fed-batch process. Traditionalfed-batch processes normally result in high lactate and ammonium levelsin the cell cultures, which are known to have inhibitory effects on cellgrowth, cell density and recombinant protein expression. Fed-batchprocesses in which glucose is slowly added to the culture have beenshown to lower lactate production and improve cell growth, cell densityand recombinant protein expression. However, prior art methods formanipulation of glucose addition are not practical for large-scalemanufacturing. Here, by utilizing culture media with lower startinglevels of glutamine and eliminating glutamine from the feed, it is shownthat lower levels of ammonium and lactate are produced, leading toincreased cell viability. Additionally, in glutamine-starved cultures,recombinant protein expression is increased and final osmolarity isreduced.

Materials and Methods

STRAINS AND MEDIA: anti-GDF-8 cells were cultured in a fed-batch mode inMedium 1 in 1 L Bioreactor.

CULTURE CONDITIONS: Cells were grown for twelve days in 1 L Bioreactors.Temperature was shifted from 37° C. to 31° C. on either day 4 or day 5depending on the cell growth. Three fed-batch processes were tested: anormal (control) process, a no glutamine feed process and a glutaminestarvation process. Pertinent details of these processes are listed inTable 8 and Table 9. TABLE 8 Fed-batch process in 1 L Bioreactors withno glutamine feed process. No glutamine feed Control process processStarting media 13 mM 13 mM Glutamine (mM) Glutamine feed 5 mM on Day 4No feed of glutamine Feed media Medium 5 (with 37.5 mM Medium 5 withoutglutamine) glutamine Feed schedule 4% daily from Day 5 4% daily from Day5 Temperature shift to Day 4 Day 5 31° C.

TABLE 9 Fed-batch process in 1 L Bioreactors with glutamine starvationprocess Control process Low glutamine process Starting media 13 mM 4 mMGlutamine (mM) Glutamine feed 5 mM on Day 4 No feed of glutamine Feedmedia Medium 5 (with 37.5 mM Medium 5 without glutamine) glutamine Feedschedule 4% daily from Day 5 4% daily from Day 5 Temperature shift toDay 4 Day 5 31° C.

SAMPLE ANALYSIS: Daily samples were taken from the cultures and wereanalyzed for cell density, cell viability, lactate, glutamine, andammonium levels. Titer of expressed anti-GDF-8 antibody was alsomeasured daily.

Results and Conclusions

FIG. 3 shows the cell density of cultures grown in either no glutaminefeed or control fed-batch conditions. In both cases, cell density wassimilar over the course of the experiment.

FIG. 4 shows percent cell viability in cultures grown in either noglutamine feed or control fed-batch conditions. The no glutamine feedculture showed a markedly higher cell viability toward the end of theexperiment, beginning on day 6.

FIG. 5 shows ammonium levels in cultures grown in either no glutaminefeed or control fed-batch conditions. The no glutamine feed cultureshowed a marked decrease in ammonium levels toward the end of theexperiment, beginning on day 4.

FIG. 6 shows lactate levels in cultures grown in either no glutaminefeed or control fed-batch conditions. Lactate levels were slightly lowerin the no glutamine feed culture throughout the course of theexperiment.

FIG. 7 shows anti-GDF-8 antibody titer in cultures grown in either noglutamine feed or control fed-batch conditions. Final anti-GDF-8antibody titer was higher in the no glutamine feed culture.

FIG. 8 shows the cell density of cultures grown in eitherglutamine-starved or control fed-batch conditions. In both cases, celldensity was similar over the course of the experiment.

FIG. 9 shows cell viability in cultures grown in eitherglutamine-starved or control fed-batch conditions. In both cases, cellviability was similar over the course of the experiment.

FIG. 10 shows ammonium levels in cultures grown in eitherglutamine-starved or control fed-batch conditions. The glutamine-starvedculture showed a marked decrease in ammonium levels throughout thecourse of the experiment.

FIG. 11 shows lactate levels in cultures grown in eitherglutamine-starved or control fed-batch conditions. The glutamine-starvedculture showed a marked decrease in lactate levels throughout towardsthe end of the experiment, beginning on day 4.

FIG. 12 shows anti-GDF-8 antibody titer in cultures grown in eitherglutamine-starved or control fed-batch conditions. Final anti-GDF-8antibody titer was higher in the glutamine-starved culture.

Collectively these results indicate that decreased glutamine levels arebeneficial to cell cultures by reducing the amount of ammoniumproduction, increasing cell viability and increasing titer of expressedanti-GDF-8 antibody. In addition, in the glutamine-starved cultures, lowlactate levels were observed, possibly due to the decreased glucoseconsumption rate. Decreased ammonium and lactate levels also have theeffect of reducing total osmolarity. Elevated osmolarity is also knownto have inhibitory effects on cell growth and viability. Low initialglutamine levels together with the elimination of the glutamine feedalso has the positive effect of reducing ammonium produced as a resultof non-enzymatic glutamine degradation in stored media. Elimination ofglutamine in the feed also simplifies the process of culturinganti-GDF-8 cells.

Example 4 Iron Dose Response of Anti-GDF-8 Cells in Medium 1 and Medium2

Medium 1 is much more concentrated in nutrients than Medium 2. Theoptimum iron levels for cell growth in Medium 1 were determined in orderto avoid problems with iron deficiency during cell culture.

Materials and Methods

Anti-GDF-8 cells were cultured in dishes for one passage in eitherMedium 1 or Medium 2. Iron concentrations of these media weremanipulated by addition of different amounts of stock iron solution.Final cell densities were measured by CEDEX.

Results and Conclusions

FIG. 13 shows the Fe dose response of anti-GDF-8 cells in Medium 1 andMedium 2 containing different iron concentrations. In Medium 2, the celldensity was relatively constant for iron concentrations ranging from 3μM to 15 μM. In Medium 1, cell density increases with increasing ironconcentration but reaches a maximum after approximately 5 μM. Thisdifference could be due to the high nutrient content in Medium 1, whichmight reduce iron availability to the cells as a consequence ofchelation of iron in the media. These results indicate that iron levelsshould be kept above 5 μM to avoid problems with iron deficiency inMedium 1.

Example 5 Substitution of Glutamate for Glutamine in the BioreactorProcess

Three experiments were performed to test the effects of substitutingglutamate for glutamine in an anti-Lewis Y cell culture process.

Materials and Methods

The experiments were performed in 10 L bioreactors at pH 7.1, 30%dissolved oxygen, and a starting temperature of 37° C. with a shift to31° C. on day 5. Sparge and headspace gasses were 88% of a 93% air/7%CO₂ mix and 12% oxygen. The starting media in all experiments was Medium1, which contains glutamine. Feed media and feed schedule includingsupplemental glucose and glutamine feeds are shown in Table 10. Columnslabeled “Glutamate” were fed with modified Medium 5, containing noglutamine, but containing a molar concentration of glutamate equal tothe molar glutamine concentration in standard Medium 5. Columns labeledglutamine were fed with standard Medium 5. TABLE 10 Feed schedule.9040-44 9040-56 9040-64 Day Glutamate 1 Glutamine 1 Glutamate 2Glutamine 2 Glutamate 3 Glutamine 3 0 1 2 3 4 5 mM gln 5 mM gln 3 g/Lgluc3 7.7 mM gln 2.9 g/l gluc 5 3.6 g/gluc 5 mM gln 3.5 g/L gluc 5 mMgln 3 g/L gluc 3 g/L gluc 5.5 g/L gluc 6 g/L gluc 6 12% 12% 17% 17% 29%29% 16XMedium 4 16XMedium 4 Medium 5 16XMedium 4 Medium 5 Medium 5 7 4mM gln 8 9 2.5 g/L gluc 10 10% 10% 8% Medium 5 5% 16XMedium 4 16XMedium4 16XMedium 4 11 1 g/L gluc 12 13 1 g/L glucResults and Conclusions

Within each experiment, cell density is similar as shown in FIG. 14.Cell densities are low in the Glutamine 2 and Glutamate 2 experimentsdue to a pH deviation to about 6.7 on day 3 on the process. The drop indensity between day 6 and 7 in the Glutamine 3 and Glutamate 3experiments is due to the 29% media feed on day 6.

FIG. 15 shows cell viability of the glutamate and glutamine fedcultures. Viabilities remained higher during the second half of theprocess in the bioreactors containing glutamate fed cultures.

In Experiment 1, anti-Lewis Y titer is similar between the glutamate andglutamine fed cultures. FIG. 16 shows that in Experiments 2 and 3,anti-Lewis Y titers are lower in the glutamine fed reactors. The loweranti-Lewis Y titer observed in these reactors could be due to the highlevels of lactate produced, as shown in FIG. 17.

Bioreactors run with glutamate in the feed media have a lower ammoniumconcentration (FIG. 18) and a lower osmolarity (FIG. 19).

The binding ELISA assay was used to test activity of samples from theGlutamine 1 and Glutamate 1 experiments. The activities were similar:110% of reference for the Glutamine 1 sample and 122% of reference forthe Glutamate 1 sample (data not shown).

The substitution of glutamate for glutamine in these experiments doesnot have a significant effect on cell density. However, cell viabilityis lower in the Bioreactors fed with glutamine. Ammonium, lactate andosmolarity are lower in the Bioreactors fed with glutamate compared tothose fed with glutamine. On average, anti-Lewis Y titer is higher inthe Bioreactors fed with glutamate and activity is essentially the sameunder both conditions.

Example 6 Substitution of Glucose and Glutamine in the Anti-Lewis Y CellCulture Process

The purpose of this experiment was to test the effects of substitutionof glucose and glutamine with the feed media listed in Table 11 below inthe culturing of anti-Lewis Y cells (see Bogheart et al.,Antibody-targeted chemotherapy with the calicheamicin conjugatehu3S193-N-acetyl gamma calicheamicin dimethyl hydrazide targets Lewisyand eliminates Lewisy-positive human carcinoma cells and xenografts,Clin. Can. Res. 10:4538-49 (2004)). Cell density, cell viability,anti-Lewis Y titer and ammonium levels were measured.

Materials and Methods

The experiment was performed in 250 ml shake flasks at a starting volumeof 75 ml. All shake flasks were seeded at 0.25×10⁶ cells/ml in Medium 2.The flasks were incubated at 37° C. in a 7% CO₂ incubator for 14 days.On days 3 and 4, the flasks were fed with 5% by volume of Medium 6 feedmedium. The composition of Medium 6 is listed in Table 11. On days 5-13the flasks were fed with 5% by volume of one of the feed solutionslisted in Table 12. Each condition was performed in duplicate. Sampleswere taken daily for cell counts by CEDEX and assays for ammonium,glucose, and lactate TABLE 11 Composition of Medium 6. Amino Acids mg/LmM alanine 142.48 1.60 arginine 1528.84 8.79 asparagine.H₂O 1080.60 7.20aspartic acid 532.40 4.00 cystine.2HCl 473.00 1.51 glutamic acid 235.381.60 glutamine 4820.00 33.01 glycine 120.07 1.60 histidine.HCl.H₂O588.32 2.80 isoleucine 944.52 7.21 leucine 1360.75 10.39 lysine.HCl1456.80 8.00 methionine 477.06 3.20 phenylalanine 660.36 4.00 proline552.31 4.80 serine 1264.70 12.04 threonine 762.02 6.40 tryptophan 260.941.28 tyrosine.2Na.2H₂O 832.62 3.19 valine 749.21 6.40 Vitamins mg/L mMbiotin 3.28 0.01 calcium pantothenate 36.02 0.08 choline chloride 143.281.03 folic acid 42.43 0.10 inositol 201.71 1.12 nicotinamide 32.02 0.26pyridoxine.HCl 32.82 0.16 riboflavin 3.60 0.01 thiamine.HCl 35.22 0.10vitamin B12 11.21 0.01 Inorganic Salts mg/L mM KH₂PO₄ 1635.00 12.02MgSO₄.7H₂O 171.98 0.70 Trace Elements μg/L nM Sodium Selenite 40.00231.35 CuSO₄ 3.44 21.51 CuSO₄.5H₂O 7.49 30.00 FeSO₄.7H₂O 2534 9115ZnSO₄.7H₂O 2704 9421 MnSO₄.H₂O 0.17 1.01 Na₂SiO₃.9H₂O 140 492.84(NH₄)₆Mo₇O₂₄.4H₂O 1.24 1.00 NH₄VO₃ 0.65 5.56 NiSO₄.6H₂O 0.13 0.49SnCl₂.2H₂O 0.12 0.53 AlCl₃.6H₂O 1.20 4.97 AgNO₃ 0.17 1.00 Ba(C₂H₃O₂)₂2.55 9.98 KBr 0.12 1.01 CdCl₂.2.5H₂O 2.28 9.99 CoCl₂.6H₂O 2.38 10.00CrCl₃ 0.32 2.02 NaF 4.20 100.02 GeO₂ 0.53 5.07 KI 0.17 1.02 RbCl 1.2110.01 ZrOCl₂.8H₂O 3.22 9.99 Other Components μg/L nM Hydrocortisone 2880.79 Putrescine.2HCl 8000 49.66 linoleic acid 336.25 1.20 thioctic acid840.63 4.08 Other Components mg/L mM D-glucose 33005.99 183.37(Dextrose) PVA 2400.00 Nucellin ™ 80.00

TABLE 12 Feeds on days 5-13. Modified Medium 6 contains no glucose orglutamine. GluGln Modified Medium 6 + 43 g/L glucose + 4.82 g/Lglutamine (control) Glu Modified Medium 6 + 43 g/L glucose + 4.82 g/Lglutamate GluAsp Modified Medium 6 + 43 g/L glucose + 4.36 g/Lasparagine GluGlyGln Modified Medium 6 + 43 g/L glucose + 6.71 g/Lglycylglutamine GluGlu Modified Medium 6 + 43 g/L galactose + 4.82 g/Lglutamine GalGlu Modified Medium 6 + 43 g/L galactose + 4.82 g/Lglutamate GalGln Modified Medium 6 + 43 g/L galactose + 4.36 g/Lasparagine GalGlyGln Modified Medium 6 + 43 g/L galactose + 6.71 g/Lglycylglutamine GalAsp Medium 6 + 43 g/L glucoseResults and Conclusions

The highest cell density was seen when glutamate or glycylglutamine wassubstituted for glutamine in the presence of either glucose or galactosein the feed media. Cell density was generally lower in the cultures fedwith glucose/glutamine, galactose/glutamine, or glucose only (FIG. 20).Final viability was highest in the cultures fed with glucose only,followed by the cultures fed with glucose/glutamate. The lowestviability was seen in the cultures fed with glutamine or asparaginecombined with either glucose or galactose (FIG. 21).

Day 14 titer was highest in the glucose/glycylglutamine and theglucose/glutamate fed cultures at about 700 μg/ml. Titer was lowest inthe galactose/glycylglutamine and the galactose/asparagine fed culturesat about 500 μg/ml. Titer in the glucose/glutamine control was about 570μg/ml (FIG. 22).

The lowest ammonium levels were seen in the flasks fed withglucose/glutamate or glucose only. The flasks fed withgalactose/glutamate, glucose/glutamine, glucose/glycylglutamine, andglucose/asparagine showed intermediate levels of ammonium. The flasksfed with galactose/asparagine, galactose/glycylglutamine, andgalactose/glutamine had the highest levels of ammonium (FIG. 23).

Glucose levels remained above 1 g/L in all flasks fed with galactoseuntil day 11. From day 11 through day 14, the glucose in these cultureswas never completely depleted, remaining between 0.6 and 1 g/L, with nosignificant differences between the different cultures.

Glucose levels increased in all flasks fed with glucose or glucosecombined with another substrate until day 10. From day 10 through day 14in these cultures, glucose levels remained fairly constant and similarto each other. On day 14 about 8.4 g/L glucose remained in theglucose/glutamate fed cultures and about 10.8 g/L glucose remained inthe cultures fed with glucose only.

Lactate levels reached a high of about 2.4 g/L on day 5, when conditionswere the same for all cells, and dropped to essentially zero in allcultures by day 14. Lactate levels were highest from day 10 through day14 in the glucose/glutamine control, but were below 1 g/L during thistime (data not shown).

All conditions tested in this experiment resulted in higher cell densitythan the control glucose/glutamine condition. All conditions testedexcept the galactose/asparagine condition resulted in higher finalviability than either the glucose/glutamine control or thegalactose/glutamine fed condition. Titer in the glucose/glutaminecontrol was about 570 μg/ml compared to a high of about 700 μg/ml in theglucose/glycylglutamine fed condition and the glucose/glutamate fedcondition.

Example 7 Evaluation of a Glutamine Starved Batch Process for theProduction of Anti-GDF-8

Typical fed-batch production methods require multiple feeds over theculture period. These feeds are designed to replace nutrients in themedium that may have been depleted by the cells or may have degradedduring the batch. These feeds create complications when the process isscaled up to be used in larger reactors, such as the need for animpeller jump (see FIG. 24). Furthermore, the feeds dilute the amount ofanti-GDF-8 already secreted into the culture and therefore affect theharvest titer. The use of a batch process would allow inoculation of thebioreactor at full volume, instead of at a partial volume so as toaccommodate the feeds, which would remove the necessity of an impellerjump and greatly reduce any dilution effect on productivity.

Glutamine is one of the most important reasons that a fed-batch approachis used since it is not stable at 37° C. and it had been thought that itneeded to be replenished during a batch culture. However, results ofExamples 2, 5, and 6, in which a glutamine starvation strategy wastested, showed a significant increase in productivity compared to acontrol reactor that was fed glutamine. This result was combined withthe batch process to create a glutamine starvation batch process thatwas tested in this Example.

Materials and Methods

Anti-GDF-8 cells were grown in 1 L Bioreactors for 12 days according tothe following four growth conditions. Bioreactor parameters for allconditions were kept the same. Dissolved oxygen was maintained at nolower than 23% of air saturation by sparging with air and pH wasmaintained at 7.00 by the addition of a solution containing sodiumbicarbonate at 0.58 M and sodium carbonate at 0.71 M. The temperature ofall cultures was maintained at 37° C. for the first four days of thebatch. On the fourth day of the batch the temperature of all thebioreactors was lowered to 31° C. and maintained at this point for theduration of the batch. The control and fed-batch cultures were fed with8%, 12%, and 8% total reactor volume of their respective feed media ondays 5, 7, and 10, respectively.

1) Control.

-   -   Inoculation medium Medium 7 (see Table 13).    -   Feed Medium 8, fed on days 5, 7, and 10 (see Table 13).    -   Feed 5 mM of glutamine on day 4.    -   Lower the temperature to 31° C. on day 4.

2) Fed-Batch Glutamine Starvation.

-   -   Inoculation medium Medium 7 with only 4 mM of glutamine (see        Table 13).    -   Feed Medium 8 without glutamine, fed on days 5, 7, and 10 (see        Table 13).    -   No glutamine feed on day 4.    -   Lower the temperature to 31° C. on day 4.

3) Batch Glutamine Starvation.

-   -   Inoculation medium new batch medium with only 4 mM of glutamine        (see Table 13).    -   No feed medium.    -   No glutamine feed.    -   Lower the temperature to 31° C. on day 4.    -   Add 5 g/L of glucose on day 8.

4) Batch Glutamine Starvation Supplemented on Day 8.

-   -   Inoculation medium new batch medium with only 4 mM of glutamine        (see Table 13).    -   No feed medium.    -   No glutamine feed.    -   Lower the temperature to 31° C. on day 4.

Add 4 g of glucose, 375 mg of Asparagine, 3 mL of 1 mM FeSO₄ stock, 3.33mL of 5 g/L Nucellin™ stock, 2.57 mL of 36 mg/L Hydrocortisone and 1.0g/L Putrescine stock solution, 0.23 mL of 50 mg/L Sodium Selenite stock,and 13.1 mg of Thiamine on day 8. TABLE 13 Compositions of media used.MW Medium 7 Medium 8 Batch media Amino acids mM mM mM L-Alanine 89.01.08 2.4 0.2 L-Arginine 174.0 6.84 13.2 4 L-Asparagine.H₂O 150.0 4.7621.4 7.5 L-Aspartic acid 133.0 2.40 6 1.65 L-Cysteine.HCl.H₂O 176.0 0.400 0.4 L-Cystine.2HCl 313 0.95 1.875 1 L-Glutamic acid 147.0 1.08 2.41.08 L-Glutamine 146.0 13.00 37.5 4 Glycine 75.0 1.28 2.4 1.54L-Histidine.HCl.H₂O 210.0 1.76 4.2 1.76 L-isoleucine 131.0 4.76 10.82.83 L-leucine 131.0 6.52 15.6 4.7 Lysine.HCl 182.0 5.20 12 5.2L-Methionine 149.0 1.96 4.8 2.6 L-Phenylalanine 165.0 2.60 6 2.2L-proline 115.0 3.24 7.2 4.1 L-serine 105.0 8.60 18 8.6 L-threonine119.0 4.32 9.6 3.2 L-tryptophan 204.0 0.78 1.92 1.04 L-tyrosine2Na.2H₂O261.0 2.16 4.8 1.75 L-valine 117.0 4.32 9.6 4 uM uM uM Vitamins Biotin244.0 8.31 20.4 11 D-Calcium 476.0 46.06 112.8 46.06 pantothenatecholine chloride 139.0 632.2 1548 840 folic acid 441.0 58.8 144 58.8I-inositol 180.0 686 1680 911 nicotinamide 122.0 161.7 396 215pyridoxine.HCl 206.0 88.15 240 88 pyridoxal.HCl 203.0 10 0 10 riboflavin376.0 5.37 13.2 1.1 thiamine.HCl 337.0 63.7 274.7 117 vitamin B12 1355.04.9 12 7.8 Inorganic salts NaCl 58.5 18.8 mM KCl 74.6 4.2 mM 4.19 mMCaCl₂ 111 1.05 mM 1.05 mM Sodium Selenite 173 27 ug/L 60 ug/L 60 ug/LNaH₂PO₄.H₂O 142 4.68 mM 11 mM 4.68 mM Na₂HPO₄ 138 0.5 mM 0.3986 mM MgSO₄120 1.15 mM 1.05 mM 1.15 mM MgCl₂ 95 0.3 mM 0.3 mM FeSO₄.7H₂O 278 9 uM24.675 uM 9 uM Fe(NO₃)₃.9H₂O 404 0.125 uM 0.124 um ZnSO₄.7H₂O 287 9.2 uM17 uM 9.2 um CuSO₄ 160 0.05 uM 0.074 uM 0.064 um NaHCO₃ 84 23.8 mM 23.8mM Others Glucose 180 16 g/L 38.3 g/L 15 g/L Polyvinyl alcohol 2.56 g/L2.4 g/L 2.56 g/L Hydrocortisone 363 0.23 mg/L 0.43 mg/L 0.28 mg/LPutrescine.2HCl 161 6.4 mg/L 12 mg/L 7.7 mg/L Sodium pyruvate 110 500 uM500 uM linoleic acid 280 0.81 uM 1.8 uM 0.81 uM thioctic acid 206 2.7 uM6 uM 2.7 uM Nucellin ™ 54 mg/L 120 mg/L 50 mg/L 1000x Trace B 1.5 m/L6.75 m/L 1.5 m/LResults and Conclusions

Cell growth for the first 4 days was similar for the control and batchprocesses, while the glutamine starved fed-batch process had a slightlylower cell density and remained a little lower for the rest of thebatch. Both batch processes maintained higher cell densities for theduration of the batch, probably due to the lack of any significantdilution (see FIG. 25). Viabilities of all the cultures were the same upto day 8. However, it is interesting to note that on day 11, theviability of the batch process that was not supplemented was lower thanthe other three bioreactors and ended up significantly lower by thefinal day. This suggests that the batch medium could still be optimizedsince the supplemented batch process had a viability that was the sameas the fed-batch bioreactors (see FIG. 26).

Cells cultured in either glutamine starved batch process or in theglutamine starved fed-batch process outperformed the same cells culturedin the control fed-batch process in productivity. The control fed-batchprocess had a harvest day titer of 685 μg/mL, as expected, while theglutamine starved fed-batch process had a harvest titer of 1080 μg/mL,about 58% higher than the control. This is similar to results seenpreviously. The glutamine starved non-supplemented batch process had aharvest day titer of 960 μg/mL, 40% higher than the control, similar tothe glutamine starved fed-batch process, while the supplementedglutamine starved batch process had the highest titer at 1296 μg/mL.This is an 89% increase over the control (see FIG. 27).

When the inhibitor levels for the four conditions were analyzed theresults showed that the lactate and ammonia levels for all threeglutamine starved processes were significantly lower than the control.In fact, after day 4, those three conditions either stopped producing orstarted consuming lactate while the control continued to produce lactatethroughout the batch (see FIG. 28). As expected, the ammonia levels weremuch lower in the glutamine starved processes and declined after day 4,while the control continued to produce ammonia (see FIG. 29).

In this Example, combining a batch process with a glutamine starvationstrategy resulted in a 40% improvement in productivity over the controlfed-batch process for anti-GDF-8 cells. The data also suggest that withsome optimization of the batch medium, an almost 2-fold improvement inproductivity can be attained. This improvement in productivity can beattributed to two factors. First, glutamine starvation increasesproductivity either directly or by keeping ammonia and lactate levelsvery low. Second, because of the absence of feeds, the titer is notdiluted during the batch. Increased productivity together with the easeof operation inherent in a batch process makes this an attractive optionfor producing recombinant polypeptides.

Example 8 Effects of Glutamine and Asparagine Concentrations in BatchMedia on Anti-GDF-8 Cell Culture Process

In Examples 2, 5 and 6, it was demonstrated that glutamine starvationconferred benefits on fed-batch cultures in two cell lines, includingincreased cell growth, cell viability and titer as well as decreasedproduction of lactate and ammonium. Asparagine also seems to play a rolein batch media.

Materials and Methods

Anti-GDF-8 cells were cultured for twelve days in 1 L Bioreactors inmodified Medium 9 with differing concentrations of glutamine andasparagine. Base Medium 9 composition is listed in Table 14.Experimental variations on this base composition are listed in Table 15.The cultures were incubated at 37° C. for the first 5 days with theexception of Reactor 4, whose temperature was 30° C. for the first daydue to temperature control problems. The cultures were shifted to 31° C.on day 6. On day 7, the cultures were fed once with 5% by volume Medium5 lacking glutamine. Cultures were measured daily for cell density,anti-GDF-8 titer, lactate and ammonium levels. TABLE 14 Composition ofMedium 9. Amino Acids mg/L mM alanine 17.80 0.20 arginine 696.00 4.00asparagine.H₂O 3000.00 20.00 aspartic acid 219.45 1.65 cysteine.HCl.H₂O70.40 0.40 cysteine.2HCl 468.75 1.50 monosodium 33.80 0.20 glutamateglutamine 584.00 4.00 glycine 115.50 1.54 histidine.HCl.H₂O 474.60 2.26isoleucine 570.73 4.36 leucine 1030.70 7.87 lysine.HCl 1401.40 7.70methionine 387.40 2.60 phenylalanine 507.00 3.07 proline 539.50 4.69serine 1052.00 10.02 threonine 564.80 4.75 tryptophan 274.16 1.34tyrosine.2Na.2H₂O 745.75 2.86 valine 749.00 6.40 Vitamins mg/L mM biotin2.68 0.01 calcium pantothenate 21.92 0.05 choline chloride 158.46 1.14folic acid 25.93 0.06 inositol 163.98 0.91 nicotinamide 26.23 0.22pyridoxal.HCl 2.03 0.01 pyridoxine.HCl 36.13 0.18 riboflavin 2.41 0.01thiamine.HCl 39.43 0.12 vitamin B12 21.17 0.02 Inorganic Salts mg/L mMCaCl₂ 116.55 1.05 KCl 312.90 4.19 Na₂HPO₄ 56.60 0.40 NaCl 1100.00 18.80NaH₂PO₄.H₂O 645.84 4.68 MgSO₄ 138.00 1.15 MgCl₂ 28.50 0.30 NaHCO₃2000.00 23.81 Trace Elements μg/L nM Sodium Selenite 69.16 400.00Fe(NO₃)₃.9H₂O 50.00 123.76 CuSO₄ 10.24 64.00 CuSO₄.5H₂O 99.88 400.00FeSO₄.7H₂O 4170 15000 ZnSO₄.7H₂O 2640 9200 MnSO₄.H₂O 33.80 200.00Na₂SiO₃.9H₂O 284.07 1000 (NH₄)₆Mo₇O₂₄.4H₂O 247.20 200.00 NH₄VO₃ 2.3420.00 NiSO₄.6H₂O 5.26 20.00 SnCl₂.2H₂O 0.90 4.00 AlCl₃.6H₂O 0.97 4.00KBr 0.48 4.00 CrCl₃ 15.83 100.00 NaF 0.17 4.00 GeO₂ 0.42 4.00 KI 33.20200.00 RbCl 0.48 4.00 H₃BO₃ 12.37 200.00 LiCl 0.17 4.00 Other Componentsμg/L nM Hydrocortisone 540.00 1.49 Putrescine.2HCl 15000 93.11 linoleicacid 290.00 1.04 thioctic acid 716.00 3.48 Other Components mg/L mMD-glucose (Dextrose) 15000.00 83.33 PVA 2560.00 Nucellin ™ 50.00 SodiumPyruvate 55.00 0.50

TABLE 15 Glutamine and Asparagine conditions tested. Reactor ReactorReactor Reactor Reactor 1 2 3 4 5 Reactor 6 Cell line anti-GDF-8 MediaBatch media (Medium 9) Glutamine 1 mM  1 mM  1 mM 4 mM  4 mM  4 mMlevels Asparagine 8 mM 12 mM 20 mM 8 mM 12 mM 20 mM levels Seeding 0.3to 0.35 density (×10⁶/ml) Feed media Medium 5-Glutamine, 5% on Day 7Culture Days 12 Temperature Day 6 Day 6 Day 6 Day 5 Day 5 Day 4 shift(37-31° C.)Results and Conclusions

FIGS. 30, 31, 32 and 33 show the cell growth of anti-GDF-8 cells,anti-GDF-8 titer, lactate levels and ammonium levels, respectively,throughout the course of the experiments under the various experimentalconditions.

Under all experimental conditions, 4 mM glutamine is better than 1 mMglutamine at all the Asparagine levels tested. At comparable glutaminelevels, 12 mM and 20 mM asparagine conditions are better than 8 mMasparagine conditions. Decreased lactate and NH₄ levels were observed atthe end of the culture for all conditions tested.

Example 9 Effects of Glutamine and Asparagine Concentrations in BatchMedia on Anti-GDF-8 Cell Culture Process

In Example 8, it was demonstrated that Medium 9 containing an initialconcentration of 4 mM glutamine performs better than media containing 1mM glutamine, regardless of asparagine levels. This example demonstratesthe effect of media containing 13 mM glutamine levels and variousasparagine levels.

Materials and Methods

Anti-GDF-8 cells were cultured for twelve days in 1 L Bioreactors inmodified Medium 9 with differing concentrations of glutamine andasparagine as listed in Table 16. The cultures were incubated at 37° C.for the first 3 days. The cultures were then shifted to 31° C. on day 4.On day 7, the cultures were fed once with 5% by volume Medium 5 lackingglutamine. Cultures were measured periodically for cell density, cellviability, lactate, ammonium levels and glutamine levels, anti-GDF-8titer, and osmolarity. TABLE 16 Glutamine and Asparagine conditionstested. Reactor Reactor Reactor Reactor Reactor 1 2 3 4 5 Reactor 6 Cellline anti-GDF-8 Media Batch media (Medium 9) Glutamine  4 mM  4 mM 13 mM13 mM 13 mM 13 mM levels Asparagine 20 mM 20 mM 20 mM 12 mM 12 mM  8 mMlevels Seeding 0.3 to 0.35 density (×10⁶/ml) Feed media Medium 5 lackingglutamine, 5% on Day 7 Culture Days 12 Temperature Day 4 Day 4 Day 4 Day4 Day 4 Day 4 shift (37-31° C.)Results and Conclusions

FIGS. 34, 35, 36, 37, 38, 39 and 40 show the cell growth of anti-GDF-8cells, percent viability of anti-GDF-8 cells, lactate levels, ammoniumlevels, glutamine levels, anti-GDF-8 titer, and osmolarity,respectively, throughout the course of the experiments under the variousexperimental conditions.

Among all the conditions tested, only Medium 9 containing 13 mMglutamine and 20 mM asparagine showed significant adverse effects oncell growth and titer. Glutamine is exhausted in all the cultures atapproximately the same time, regardless of whether the culture beginswith 4 mM or 13 mM glutamine. The highest anti-GDF-8 titer is obtainedin cultures that contain 13 mM glutamine and 12 mM asparagine. Allculture conditions exhibit decreased lactate and ammonium levels nearthe end of the culture. Ammonium levels were highest in the culturecontaining 13 mM glutamine and 20 mM asparagine.

Example 10 The Effect of Asparagine and Cysteine Levels on the ObservedDecrease in Lactate and Ammonium Levels in Anti-GDF-8 Cells Cultured inMedium 9

In Examples 2, 5 and 6, it was found that cultures grown under glutaminestarvation conditions exhibit decreased lactate and ammonium levels atthe end of the culture process. However, cultures grown in Medium 9under non-glutamine starvation conditions still exhibit decreasedlactate and ammonium levels at the end of the culture process. Thiseffect was not observed in other media such as Medium 1, where glutaminestarvation appears necessary for the decreased levels of lactate andammonium. Medium 9 and Medium 1 differ in the levels of asparagine (20mM in Medium 9 versus 11 mM total in Medium 1 plus feed) and acidiccystine (1.5 mM in Medium 9 versus 0.95 mM in Medium 1). This exampletests whether these two components were responsible for the observeddecrease in the lactate and ammonium levels at the end of the culture.

Materials and Methods

Anti-GDF-8 cells were cultured in 1 L BioReactors for 12 days. Cellswere initially cultured at 37° C. and were shifted to 31° C. on day 4 orday 5 at 8-10×10⁶/ml. Table 17 lists the various experimental conditionstested. Samples were taken daily and saved for titer analysis by ProteinA HPLC. TABLE 17 Asparagine and cysteine conditions tested. Media Gln(mM) Asn (mM) Total Gln (mM) Total Asn (mM) Feed Medium 1 13 5 29 11Medium 5, 30% 5 mM Gln, day 4 total Medium 1 4 5 4 11 Medium 5-Gln, 30%total Batch media (Medium 9) 4 20 4 21 Medium 5-Gln, 5% day 7 Medium 1 +5 mM Asn + 0.5 mM 13 10 29 16 Medium 5, 30% 5 mM Gln, day 4 Cysteinetotal Batch media (Medium 9) 13 20 29 21 Medium 5, 30% 5 mM Gln, day 4totalNote:Medium 5-Gln = Medium 5 lacking glutamine.Results and Conclusions

Anti-GDF-8 cells grown in Medium 9 exhibited decreased lactate andammonium levels at the end of the culture process, regardless of whetherthe cultures were started with 4 mM or 13 mM glutamine (see FIGS. 42 and43). In contrast, Medium 1 only exhibited decreased lactate and ammoniumlevels at the end of the culture process when the cultures were startedwith 4 mM glutamine (see FIGS. 42 and 43). Addition of extra asparagineand cystine to Medium 1 containing 13 mM glutamine did not result indecreased lactate and ammonium levels at the end of the culture process(see FIGS. 42 and 43).

Cultures that exhibited decreased lactate and ammonium levels at the endof the culture process (Medium 1 with 4 mM glutamine, Medium 9 with 4 mMglutamine and Medium 9 with 13 mM glutamine) were also observed to havelower total osmolarity at the end of the culture process (see FIG. 47).

Medium 9 with 4 mM glutamine exhibited the highest anti-GDF-8 titer,followed by Medium 9 with 13 mM glutamine fed on day 4 (see FIG. 46).Taking the effect of dilution of the feed into account, Medium 9containing 4 mM glutamine had equivalent anti-GDF-8 titer to Medium 9containing 13 mM glutamine.

Example 11 The Effect of Amino Acid and Vitamin Levels on the ObservedDecrease in Lactate and Ammonium Levels in Anti-GDF-8 Cells Cultured inMedium 9

Example 10 tested whether difference in the asparagine and cysteinelevels between Medium 1 and Medium 9 were responsible for the observeddecrease in lactate and ammonium levels at the end of the cultureprocess in Medium 9 that was not starved for glutamine. It wasdetermined that these factors were not responsible for the observeddecrease. Medium 1 and Medium 9 also differ in their amino acid andvitamin concentrations. This example tests whether differences in aminoacids and vitamin concentrations between these two media are responsiblefor the observed decrease.

Materials and Methods

Anti-GDF-8 cells were cultured in 1 L BioReactors for 12 days. Cellswere initially cultured at 37° C. and were shifted to 31° C. on day 4 at8-10×10⁶/ml. Table 18 lists the various experimental conditions tested.Amino acids, vitamins, hydrocortisone and putrescine, trace elements E(composition listed in Table 19) and iron were added to the variousexperimental Medium 1 conditions such that the levels of thesecomponents were equal to the levels in Medium 9. Samples were takendaily and saved for titer analysis by Protein A HPLC. TABLE 18 Aminoacid and vitamin conditions tested. Gln Asn Media (mM) (mM) Feed Day 5Day 7 Day 10 Day 11 Medium 1 13 5 Medium 5 30% total 5 mM Gln, day 4 8%Medium 5 12% 8% Medium 5 Medium 5 Medium 1 + AA 13 15 Medium 5 30% total5 mM Gln, day 4 8% Medium 5 12% 8% Medium 5 Medium 5 Medium 13 5 Medium5 30% total 5 mM Gln, day 4 8% Medium 5 12% 8% 4 g/L glucose 1 + Vit,H/P, E, Fe Medium 5 Medium 5 Medium 1 + all 13 15 Medium 5 30% total 5mM Gln, day 4 8% Medium 5 12% 8% 4 g/L glucose Medium 5 Medium 5 Medium9 with 13 mM 13 20 Medium 5 30% total 5 mM Gln, day 4 8% Medium 5 12% 8%Gln Medium 5 Medium 5Note:AA = Amino acids,H/P: = 0.036 mg/mL hydrocortisone, 1.08 mg/mL Putrescine.2HCl,E: Trace Elements E.

TABLE 19 Composition of Trace Elements E. Trace Elements μg/L nM(NH₄)6Mo₇O₂₄.4H₂O 123.60 100.00 AlCl₃.6H₂O 0.48 2.00 H₃BO₃ 6.18 100.00CrCl₃ 7.92 50.00 CuSO₄.5H₂O 49.94 200.00 GeO₂ 0.21 2.00 KBr 0.24 2.00 Kl16.60 100.00 LiCl 0.08 2.00 MnSO₄.H₂O 16.90 100.00 Na₂SiO₃.9H₂O 142.03500.00 NaF 0.08 2.00 NH₄VO₃ 1.17 10.00 NiSO₄.6H₂O 2.63 10.00 RbCl 0.242.00 SnCl₂.2H₂O 0.45 2.00 Sodium Selenite 34.58 200.00Results and Conclusions

All conditions tested exhibited decreased lactate and ammonium levels atthe end of the culture process except for Medium 1 containing addedamino acids, indicating that increased amino acid levels in Medium 9compared to Medium 1 are probably not responsible for the decreases inlactate and ammonium levels (see FIGS. 49 and 50). However, Medium 1containing added vitamins, hydrocortisone and putrescine, trace elementsE and iron exhibited lower lactate and ammonium levels at the end of theculture process compared to Medium 1 containing added amino acids (seeFIGS. 49 and 50). This indicates that these components may beresponsible for the observed decreases in Medium 9.

Cultures grown in Medium 1 containing added vitamins, hydrocortisone andputrescine, trace elements E and iron exhibited the lowest levels ofammonium throughout the experiment due to the lower total amounts ofasparagine and glutamine in the starting media (see FIG. 50).

Example 12 The Effect of Vitamin, Trace Elements E and Iron Levels onthe Observed Decrease in Lactate and Ammonium Levels in Anti-GDF-8 CellsCultured in Medium 9

In Example 11, it was determined that the increased levels of vitamins,hydrocortisone and putrescine, trace elements E and iron in Medium 9relative to Medium 1 might be responsible for the decrease in lactateand ammonium levels observed at the end of the culture process. Here,these components were tested individually and in combination todetermine which, if any, were responsible for the observed decrease.

Materials and Methods

Anti-GDF-8 cells were cultured in 1 L BioReactors for 12 days. Cellswere initially cultured at 37° C. and were shifted to 31° C. on day 4 at8-10×10⁶ cells/ml, with the exception of Medium 1 containing trace Eelements, which were shifted on day 4 at about 6×10⁶ cells/ml. Table 20lists the various experimental conditions tested. Hydrocortisone andputrescine were added to all Medium 1 conditions such that the levels ofthese components were equal to the levels in Medium 9. Vitamins, Traceelements E (composition listed in Table 19) and iron were added to thevarious experimental Medium 1 conditions such that the levels of thesecomponents were equal to the levels in Medium 9. Samples were takendaily and saved for titer analysis by Protein A HPLC. TABLE 20 Aminoacid and vitamin conditions tested. Gln Asn Media (mM) (mM) Feed TempShift Day 4 Day 5 Day 7 Day 10 Medium 1 + Fe 13 15 Medium 5 30% day 4 5mM Gln, day 4 8% Medium 5 12% 8% Medium 5 total Medium 5 Medium 1 + E 1315 Medium 5 30% day 4 5 mM Gln, day 4 8% Medium 5 12% 8% Medium 5 totalMedium 5 Medium 1 + Vit 13 15 Medium 5 30% day 4 5 mM Gln, day 4 8%Medium 5 12% 8% Medium 5 total Medium 5 Medium 1 + Fe + E 13 15 Medium 530% day 4 5 mM Gln, day 4 8% Medium 5 12% 8% Medium 5 total Medium 5Medium 1 + Fe + Vit 13 15 Medium 5 30% day 4 5 mM Gln, day 4 8% Medium 512% 8% Medium 5 total Medium 5 Medium 1 + E + Vit 13 15 Medium 5 30% day4 5 mM Gln, day 4 8% Medium 5 12% 8% Medium 5 total Medium 5 Medium 9with 13 mM 13 20 Medium 5 30% day 4 5 mM Gln, day 4 8% Medium 5 12% 8%Medium 5 Gln total Medium 5Note:E: Trace Elements E.Results and Conclusions

Of all the conditions tested, only Medium 9 containing 13 mM glutamineand Medium 1 containing trace elements E exhibited decreased levels oflactate and ammonium at the end of the culture process (see FIGS. 54 and55). It should be noted that the decreased levels observed for Medium 1containing trace elements E could be due to the fact that this culturewas temperature shifted when the cells were at about 6×10⁶ cells/mL.

Medium 9 containing 13 mM glutamine exhibited higher anti-GDF-8 titerthan any of the Medium 1 formulations.

Example 13 Comparison of Mediums 1, 3 and 9 on Cell Growth andAnti-GDF-8 Titer

This experiment was performed to measure the differences in cell growthand anti-GDF-8 titer using Mediums 1, 3 and 9.

Materials and Methods

Anti-GDF-8 cells were cultured in various media and under feedingconditions as listed in Table 21. Pertinent media information is listedin Table 22. Cells were grown in 1 L Bioreactors for 12 days and wereshifted from 37° C. to 31° C. on day 4. TABLE 21 Media and feedconditions tested. Feed Media Asn Gln Day 3 Day 4 Day 5 Day 6 Day 7 Day10 Day 11 Medium 3 14 mM 4 mM 3.3% Medium 3.3% Medium 3.3% Medium 3.3%Medium 10% Medium 3.3% 3.3% Medium 5-Gln 5-Gln 5-Gln 5-Gln 5-Gln Medium5-Gln 5-Gln Medium 3 14 mM 4 mM 3.3% Medium 3.3% Medium 3.3% Medium 3.3%Medium 10% Medium 3.3% 3.3% Medium 5-Gln 5-Gln 5-Gln 5-Gln 5-Gln Medium5-Gln 5-Gln Medium 1 14 mM 4 mM   8% Medium 12% Medium   8% 5-Gln 5-GlnMedium 5-Gln Medium 9 20 mM 4 mM   5% Medium 5-GlnNote:Medium 5-Gln—Medium 5 lacking glutamine.

TABLE 22 Media summary. Media Asn Gln Feed AA(Starting)/(Total)Ion/Total AA Medium 3 14 mM 4 mM   34% Medium 5-Gln   34 mM/64 mM 1.75Medium 9 20 mM 4 mM   5% Medium 5-Gln 91.4 mM/94.3 mM .72 Medium 1 14 mM4 mM 31.6% Medium 5-Gln   78 mM/96.4 mM .74Note:Medium 5-Gln—Medium 5 lacking glutamine.Results and Conclusions

Anti-GDF-8 cells cultured in Medium 9 exhibited the highest cell densityand anti-GDF-8 titer, while anti-GDF-8 cells cultured in Medium 3exhibited the lowest cell density and anti-GDF-8 titer (see FIGS. 57 and58). The fact that Medium 9 produces superior results than Medium 1indicates that it is better to provide the media components in thestarting media rather than supplying them through multiple feeds.Additionally, the fact that both Medium 1 and Medium 9 perform betterthan Medium 3 indicates that providing amino acids in concentrationsgreater than about 70 mM provide superior results than providing aminoacids in concentrations less than about 70 mM. Finally, providing aminoacids in concentrations greater than about 70 mM in the starting mediaresults in the highest cell densities and titers (compare Medium 9 vs.Medium 1).

Example 14 Statistical Analysis of Optimum Total Glutamine andAsparagine Levels in Medium 9 for Anti-GDF-8 Cell Culture in Bioreactors

Materials and Methods

Anti-GDF-8 cells were grown in 1 L Bioreactors and were shifted from 37°C. to 31° C. on the days indicated in Table 23. Final titers weresubjected to a T-test in order to determine the optimum level ofglutamine alone and the optimum level of total combined glutamine andasparagine. Table 23 summarizes some relevant experimental conditionsand end results for anti-GDF-8 cells grown in Medium 9. TABLE 23Relevant experimental conditions and end results for anti-GDF-8 cellsgrown in Medium 9. Day Media Gln (mM) Asn (mM) Shifted Feed Titer(ug/ml) Titer/1200 Total Gln Total Asn Total Gln + A

Medium 9 1 8 6 5% Medium 5-Gln 615.2 0.51 1 9 10 Medium 9 1 8 6 5%Medium 5-Gln 857.1 0.71 4 9 13 Medium 9 1 12 6 5% Medium 5-Gln 947 0.791 13 14 Medium 9 4 12 4 5% Medium 5-Gln 1184 0.99 4 13 17 Medium 9 4 204 5% Medium 5-Gln 769.6 0.64 1 21 22 Medium 9 4 8 5 5% Medium 5-Gln1262.6 1.05 13 9 22 Medium 9 4 20 4 5% Medium 5-Gln 1198 1.00 4 21 25Medium 9 4 20 4 5% Medium 5-Gln 1321.1 1.10 4 21 25 Medium 9 4 20 4 5%Medium 5-Gln 1162.4 0.97 4 21 25 Medium 9 13 20 4 5% Medium 5-Gln 1436.61.20 4 21 25 Medium 9 15 12 4 5% Medium 5-Gln 1638.6 1.37 13 13 26Medium 9 13 12 4 5% Medium 5-Gln 1606.7 1.34 13 13 26 Medium 9 13 20 45% Medium 5-Gln 1075.91 0.90 13 21 34 Medium 9 13 20 4 5% Medium 5-Gln1058.4 0.88 13 21 34 Medium 9 13 20 4 5% Medium 5-Gln 1075.91 0.90 15 2136 Medium 9 13 5 4 Asn, Gln, 5% Medium 5-Gln 974.52 0.81 28.5 11 39.5Medium 9 13 20 4 Asn, Gln, 5% Medium 5-Gln 831.81 0.69 28.5 26 54.5Medium 9 13 20 4 Medium 5, 30% total, 5 mM Gln 975.4 0.81 28.5 26 54.5day 4 Medium 9 13 20 4 Medium 5, 30% total, 5 mM Gln 973.5 0.81 28.5 2654.5 day 4Note:Medium 5-Gln—Medium 5 lacking glutamine.Results and Conclusions

FIG. 59 shows extrapolated anti-GDF-8 titers for various levels ofglutamine alone and total combined glutamine and asparagine. Table 24shows the results of a T-test comparing normalized titer of glutaminelevels between 2 and 15 mM and glutamine levels outside this range.Table 25 shows the results of a T-test comparing normalized titer ofcombined glutamine and asparagine levels between 16 and 36 mM andcombined glutamine and asparagine levels outside this range.

Both T-test results indicated significant differences in anti-GDF-8titers between the two groups that were compared. Cultures grown inMedium 9 containing between 2 and 15 mM glutamine and between 16 and 36mM combined glutamine and asparagine exhibited higher anti-GDF-8 titersthan cultures grown in media with glutamine and combined glutamine andasparagine levels that fell outside these ranges. In all experiments,asparagine levels were greater than 9 mM. TABLE 24 T-Test resultscomparing normalized titer of 2 mM < Gln < 15 mM versus Gln > 15 mM, Gln< 2 mM conditions. Normalized Titer Gln > 15, Gln < 2 2 < Gln < 15 Mean0.724649917 1.033147493 Variance 0.013326655 0.036834109 Observations 712 Pooled Variance 0.028537361 Hypothesized Mean Difference 0 df 17 tStat −3.839791986 P(T <= t) one-tail 0.000656219 t Critical one-tail1.739606432 P(T <= t) two-tail 0.001312438 t Critical two-tail2.109818524

TABLE 25 T-Test results comparing normalized titer of 16 mM < Gln + Asn< 36 mM versus Gln + Asn > 36 mM, Gln + Asn < 16 mM conditions. Asn +Gln > 36, Normalized Titer Asn + Gln < 16 16 < Asn + Gln < 36 Mean0.735066584 1.027071104 Variance 0.012061148 0.041504987 Observations 712 Pooled Variance 0.031113044 Hypothesized Mean 0 Difference df 17 tStat −3.480816823 P(T <= t) one-tail 0.001430281 t Critical one-tail1.739606432 P(T <= t) two-tail 0.002860561 t Critical two-tail2.109818524

Example 15 Effects of Medium on Cell Culture

This example investigated the performance of three cell culture mediumvariations at intermediate scale utilizing high density seed cultures.All of the media tested were expected to show improvements over thePhase 1 medium (Medium 10 fed with Medium 11 feed medium), based onsmall scale bioreactor data.

Materials and Methods

CHO cells expressing a humanized anti-Abeta peptide IgG1 monoclonalantibody (“anti-ABeta cells”) were tested in various media, as shown inTable 26 (see Basi et al., Humanized Antibodies that Recognize BetaAmyloid Peptide, WO02/46237). The pH low end set point was 7.0controlled with 0.95M Na₂CO₃+0.05M K₂CO₃, except for Phase 1, which wascontrolled with a solution containing sodium bicarbonate at 0.58 M andsodium carbonate at 0.71 M. Dissolved oxygen was controlled at 30% bysparging on demand with air, agitation was at 60 rpm, and the feedmedium was Medium 5 (with or without glutamine, as noted). All cultureswere grown at 130 L scale except for 03P49B501, which was grown at 500 Lscale. In brief, Medium 1 is enriched in all nutrients, withoutconsideration for relative uptake rates, while Medium 12 was balanced byremoving apparently unnecessary nutrients from the indiscriminatelyenriched version. The compositions of Mediums 10, 11 and 12 are listedin Table 27. TABLE 26 Initial medium, feed quantities and seed sourcesfor Pilot runs. Initial Amount Gln Seed Seed Density Batch No.Description Medium Fed fed? Source (Viables/mL) 1 Phase 1 Medium 10 38%*Yes Wave bags 0.2 × 10⁶ 2 Rich Medium, Medium 1 16% Yes Wave bags 0.2 ×10⁶ High Gln (1) 3 Rich Med, High Medium 1 16% Yes Wave bags 0.2 × 10⁶Gln (2) 4 Rich Med, Medium 1 15% No Wave bags 0.2 × 10⁶ Lower Gln 5Balanced Med, Medium 12 10% No Wave bags 0.2 × 10⁶ Low Gln (1) 6 BalMed, Low Medium 12  9% No Wave bags 0.2 × 10⁶ Gln (2) 7 Bal Med, LowMedium 12  5% No High density 2.0 × 10⁶ Gln, Dense Bioreactor Seed*The Phase 1 process was fed with Medium 12, which is not as rich asMedium 5.

TABLE 27 Compositions of Mediums 10, 11 and 12. Medium 10 Medium 11Medium 12 Amino Acids mg/L mM mg/L mM mg/L mM alanine 24.87 0.28 142.481.60 17.80 0.20 arginine 423.43 2.43 1528.84 8.79 696.00 4.00asparagine.H₂O 173.90 1.16 1080.60 7.20 1500.00 10.00 aspartic acid52.72 0.40 532.40 4.00 219.45 1.65 cysteine.HCl.H₂O 70.01 0.40 70.400.40 cysteine.2HCl 62.09 0.20 470.00 1.50 312.50 1.00 glutamic acid41.08 0.28 235.38 1.60 monosodium 33.80 0.20 glutamate glutamine 1162.407.96 6000.00 41.10 584.00 4.00 glycine 35.92 0.48 120.07 1.60 115.501.54 histidine.HCl.H₂O 75.27 0.36 588.32 2.80 369.60 1.76 isoleucine151.90 1.16 944.52 7.21 370.73 2.83 leucine 172.69 1.32 1360.75 10.39615.70 4.70 lysine.HCl 218.38 1.20 1456.80 8.00 946.40 5.20 methionine53.55 0.36 477.06 3.20 387.40 2.60 phenylalanine 98.81 0.60 660.36 4.00363.00 2.20 proline 96.40 0.84 552.31 4.80 471.50 4.10 serine 273.072.60 1264.70 12.04 903.00 8.60 threonine 132.81 1.12 762.02 6.40 380.803.20 tryptophan 28.99 0.14 260.94 1.28 212.16 1.04 tyrosine.2Na.2H₂O145.10 0.56 832.62 3.19 456.75 1.75 valine 131.17 1.12 749.21 6.40468.00 4.00 Vitamins mg/L μM mg/L μM mg/L μM biotin 0.36 1.49 3.28 13.452.68 11.00 calcium pantothenate 4.03 8.47 36.02 75.67 21.93 46.06choline chloride 16.11 115.92 143.28 1030 116.76 840.00 folic acid 4.7610.80 42.43 96.22 25.93 58.80 inositol 22.64 125.79 201.71 1120 163.98911.00 nicotinamide 3.61 29.62 32.02 262.44 26.23 215.00 pyridoxal.HCl1.99 9.83 2.03 10.00 pyridoxine.HCl 1.67 8.10 32.82 159.31 18.13 88.00riboflavin 0.40 1.06 3.60 9.58 0.41 1.10 thiamine.HCl 3.92 11.64 35.22104.51 39.43 117.00 vitamin B12 1.34 0.99 11.21 8.27 10.57 7.80Inorganic Salts mg/L mM mg/L mM mg/L mM CaCl₂ 115.78 1.04 113.27 1.02116.55 1.05 KCl 310.94 4.17 312.90 4.19 KH₂PO₄ 1640.00 12.06 Na₂HPO₄70.81 0.50 56.60 0.40 NaCl 3704.96 63.44 NaH₂PO₄.H₂O 114.53 0.83 645.844.68 MgSO₄ 48.70 0.41 138.00 1.15 MgSO₄.7H₂O 8.60 0.03 170.00 0.69 MgCl₂28.53 0.30 28.50 0.30 NaHCO₃ 1220.00 14.52 2000.00 23.81 Trace Elementsμg/L nM μg/L nM μg/L nM Sodium Selenite 7.00 40.49 40.00 231.35 53.65310.27 Fe(NO₃)₃.9H₂O 49.86 123.42 50.00 123.76 CuSO₄ 0.97 6.06 3.4421.51 10.00 62.50 CuSO₄.5H₂O 7.49 30.00 7.49 30.00 49.94 200.00FeSO₄.7H₂O 1542 5549 2534 9115 3366 12000 ZnSO₄.7H₂O 1383 4821 2704 94212640 9198 MnSO₄.H₂O 0.17 1.01 0.17 1.01 16.90 100.00 Na₂SiO₃.9H₂O 140492.84 140.00 492.84 142.03 500.00 (NH₄)₆Mo₇O₂₄.4H₂O 1.24 1.00 1.24 1.00123.60 100.00 NH₄VO₃ 0.65 5.56 0.65 5.56 1.17 10.00 NiSO₄.6H₂O 0.13 0.490.13 0.49 2.63 10.00 SnCl₂.2H₂O 0.12 0.53 0.12 0.53 0.45 2.00 AlCl₃.6H₂O1.20 4.97 0.48 2.00 AgNO₃ 0.17 1.00 Ba(C₂H₃O₂)₂ 2.55 9.98 KBr .12 1.010.24 2.00 CdCl₂.2.5H₂O 2.28 9.99 CoCl₂.6H₂O 2.38 10.00 CrCl₃ 0.32 2.027.92 50.00 NaF 4.20 100.02 0.08 2.00 GeO₂ 0.53 5.07 0.21 2.00 KI 0.171.02 16.60 100 RbCl 1.21 10.01 0.24 2.00 ZrOCl₂.8H₂O 3.22 9.99 H3BO₃6.18 100.00 LiCl 0.08 2.00 Other Components μg/L μM μg/L μM μg/L μMHydrocortisone 86.40 .24 288.00 0.79 360.00 0.99 Putrescine.2HCl 248015.39 8000 49.66 10000 62.07 linoleic acid 56.69 0.20 336.25 1.20 290.001.04 thioctic acid 141.71 0.69 840.63 4.08 716.00 3.48 Other Componentsmg/L mM mg/L mM mg/L mM D-glucose (Dextrose) 11042.24 61.35 43005.99238.92 15000.00 83.33 PVA 2520.00 2400.00 2560.00 Nucellin ™ 14.00 80.0050.00 Sodium Pyruvate 54.85 0.50 55.00 0.50Results and Conclusions

Media changes led to steady improvement through the course of theseexperiments. In terms of cell growth, viability, reduced lactate levels,reduced ammonium levels, and titer, reduced glutamine levels were betterthan elevated ones (see FIGS. 60-64) and balanced (batch) medium wasbetter than rich medium (Medium 1, see FIGS. 60-64). Cultures startedfrom high density inoculum exhibited higher final titer than didcultures started from lower density inoculums (see FIG. 64).

Unlike what was observed in small scale bioreactors, the first medium(Medium 1 with high Gln) resulted in lower titers than did the originalprocess (see FIG. 64). There also was no shift to lactate uptake afterthe temperature change (see FIG. 62). This suggests that there may besome scale sensitivity with this medium. This conclusion is supported bysmall-scale (2 L) parallel runs that were done along with theseintermediate scale experiments (data not shown). The later mediumformulations containing less glutamine were not sensitive to scale, atleast in these experiments (see FIGS. 60-65). The duplicated processes(Batches 2 and 3 and Batches 5 and 6) show very good run-to-runreproducibility (see FIGS. 60-65), increasing the confidence in all ofthe data gathered in this campaign.

Example 16 Production of TNFR-Ig Using Medium 9

Materials and Methods

CHO cells expressing a dimeric fusion protein consisting of theextracellular ligand-binding portion of the human 75 kilodalton (p75)tumor necrosis factor receptor (TNFR) linked to the Fc portion of IgG1(“TNFR-Ig cells”) were seeded at high density from a perfusionbioreactor and diluted to 3×10⁶ viable cells/ml in Medium 9 for theproduction bioreactor step.

Results and Conclusions

FIGS. 66, 67, 68, 69, 70, 71 and 72 show cell growth, cell viability,residual glucose, glutamine levels, lactate concentration, ammoniumconcentration, and relative product titer, respectively. Under the rangeof minor modifications to the process, all conditions yielded good cellgrowth, high cellular viability, and high overall final titer.

For all the conditions of this experiment, the metabolic inhibitorybyproduct lactate was either consumed, or the concentration plateaued,suggesting that lactate production was arrested. Similarly, for theinhibitory metabolite ammonium, levels rose initially, but at some timeafter the temperature shift the ammonium started to be consumed by thecells. In this Example, the TNFR-Ig cell cultures were subjected to thechemical inductants sodium butyrate, and HMBA.

Example 17 Comparison of Large and Small-Scale Culture Conditions

Materials and Methods

To determine whether the size of the culture affected relevant culturecharacteristics, anti-GDF-8 cells were grown in either small-scale 1liter bioreactors or large-scale 6000 liter bioreactors. Cells weregrown at 37° C. and shifted to 31° C. on day 4.

Results and Conclusions

As can be seen in FIGS. 73, 74, 75 and 76 (which show cell density,titer, lactate levels and ammonium levels, respectively), there were norelevant differences between the 6000 liter large-scale and 1 litersmall-scale cultures for these characteristics. Both lactate andammonium levels began to decrease after the temperature shift on day 4.This example demonstrates that the size of the culture does not affectcell density, cell viability, lactate levels and ammonium levels whenthe cultures are subjected to the same growth conditions.

1. A method of producing a polypeptide in a large-scale production cellculture comprising the steps of: providing a cell culture comprising;mammalian cells that contain a gene encoding a polypeptide of interest,which gene is expressed under condition of cell culture; and a mediumcontaining glutamine and having a medium characteristic selected fromthe group consisting of: (i) a cumulative amino acid amount per unitvolume greater than about 70 mM, (ii) a molar cumulative glutamine tocumulative asparagine ratio of less than about 2, (iii) a molarcumulative glutamine to cumulative total amino acid ratio of less thanabout 0.2, (iv) a molar cumulative inorganic ion to cumulative totalamino acid ratio between about 0.4 to 1, (v) a combined cumulativeamount of glutamine and asparagine per unit volume of greater than about16 mM, and combinations thereof; maintaining said culture in an initialgrowth phase under a first set of culture conditions for a first periodof time sufficient to allow said cells to reproduce to a viable celldensity within a range of about 20%-80% of the maximal possible viablecell density if said culture were maintained under the first set ofculture conditions; changing at least one of the culture conditions, sothat a second set of culture conditions is applied; maintaining saidculture for a second period of time under the second set of conditionsand for a second period of time so that the polypeptide accumulates inthe cell culture.
 2. A method of producing a polypeptide in alarge-scale production cell culture comprising the steps of: providing acell culture comprising; mammalian cells that contain a gene encoding apolypeptide of interest, which gene is expressed under condition of cellculture; and a medium containing a cumulative amino acid amount per unitvolume greater than about 70 mM; and said medium containing glutamine;and said medium having two medium characteristics selected from thegroup consisting of: (i) a molar cumulative glutamine to cumulativeasparagine ratio of less than about 2, (ii) a molar cumulative glutamineto cumulative total amino acid ratio of less than about 0.2, (iii) amolar cumulative inorganic ion to cumulative total amino acid ratiobetween about 0.4 to 1, (iv) a combined cumulative amount of glutamineand asparagine per unit volume of greater than about 16 mM, andcombinations thereof; maintaining said culture in an initial growthphase under a first set of culture conditions for a first period of timesufficient to allow said cells to reproduce to a viable cell densitywithin a range of about 20%-80% of the maximal possible viable celldensity if said culture were maintained under the first set of cultureconditions; changing at least one of the culture conditions, so that asecond set of culture conditions is applied; maintaining said culturefor a second period of time under the second set of conditions and for asecond period of time so that the polypeptide accumulates in the cellculture.
 3. A method of producing a polypeptide in a large-scaleproduction cell culture comprising the steps of: providing a cellculture comprising; mammalian cells that contain a gene encoding apolypeptide of interest, which gene is expressed under condition of cellculture; and a medium containing a molar cumulative glutamine tocumulative asparagine ratio of less than about 2; and said mediumcontaining glutamine; and said medium having two medium characteristicsselected from the group consisting of: (i) a medium containing acumulative amino acid amount per unit volume greater than about 70 mM,(ii) a molar cumulative glutamine to cumulative total amino acid ratioof less than about 0.2, (iii) a molar cumulative inorganic ion tocumulative total amino acid ratio between about 0.4 to 1, (iv) acombined cumulative amount of glutamine and asparagine per unit volumeof greater than about 16 mM, and combinations thereof; maintaining saidculture in an initial growth phase under a first set of cultureconditions for a first period of time sufficient to allow said cells toreproduce to a viable cell density within a range of about 20%-80% ofthe maximal possible viable cell density if said culture were maintainedunder the first set of culture conditions; changing at least one of theculture conditions, so that a second set of culture conditions isapplied; maintaining said culture for a second period of time under thesecond set of conditions and for a second period of time so that thepolypeptide accumulates in the cell culture.
 4. A method of producing apolypeptide in a large-scale production cell culture comprising thesteps of: providing a cell culture comprising; mammalian cells thatcontain a gene encoding a polypeptide of interest, which gene isexpressed under condition of cell culture; and a medium containing amolar cumulative glutamine to cumulative total amino acid ratio of lessthan about 0.2; and said medium containing glutamine; and said mediumhaving two medium characteristics selected from the group consisting of:(i) a medium containing a cumulative amino acid amount per unit volumegreater than about 70 mM, (ii) a molar cumulative glutamine tocumulative asparagine ratio of less than about 2, (iii) a molarcumulative inorganic ion to cumulative total amino acid ratio betweenabout 0.4 to 1, (iv) a combined cumulative amount of glutamine andasparagine per unit volume of greater than about 16 mM, and combinationsthereof; maintaining said culture in an initial growth phase under afirst set of culture conditions for a first period of time sufficient toallow said cells to reproduce to a viable cell density within a range ofabout 20%-80% of the maximal possible viable cell density if saidculture were maintained under the first set of culture conditions;changing at least one of the culture conditions, so that a second set ofculture conditions is applied; maintaining said culture for a secondperiod of time under the second set of conditions and for a secondperiod of time so that the polypeptide accumulates in the cell culture.5. A method of producing a polypeptide in a large-scale production cellculture comprising the steps of: providing a cell culture comprising;mammalian cells that contain a gene encoding a polypeptide of interest,which gene is expressed under condition of cell culture; and a mediumcontaining a molar cumulative inorganic ion to cumulative total aminoacid ratio between about 0.4 to 1; and said medium containing glutamine;and said medium having two medium characteristics selected from thegroup consisting of: (i) a medium containing a cumulative amino acidamount per unit volume greater than about 70 mM, (ii) a molar cumulativeglutamine to cumulative asparagine ratio of less than about 2, (iii) amolar cumulative glutamine to cumulative total amino acid ratio of lessthan about 0.2, (iv) a combined cumulative amount of glutamine andasparagine per unit volume of greater than about 16 mM, and combinationsthereof; maintaining said culture in an initial growth phase under afirst set of culture conditions for a first period of time sufficient toallow said cells to reproduce to a viable cell density within a range ofabout 20%-80% of the maximal possible viable cell density if saidculture were maintained under the first set of culture conditions;changing at least one of the culture conditions, so that a second set ofculture conditions is applied; maintaining said culture for a secondperiod of time under the second set of conditions and for a secondperiod of time so that the polypeptide accumulates in the cell culture.6. A method of producing a polypeptide in a large-scale production cellculture comprising the steps of: providing a cell culture comprising;mammalian cells that contain a gene encoding a polypeptide of interest,which gene is expressed under condition of cell culture; and a mediumcontaining a combined cumulative amount of glutamine and asparagine perunit volume of greater than about 16 mM; and said medium containingglutamine; and said medium having two medium characteristics selectedfrom the group consisting of: (i) a medium containing a cumulative aminoacid amount per unit volume greater than about 70 mM, (ii) a molarcumulative glutamine to cumulative asparagine ratio of less than about2, (iii) a molar cumulative glutamine to cumulative total amino acidratio of less than about 0.2, (iv) a molar cumulative inorganic ion tocumulative total amino acid ratio between about 0.4 to 1, andcombinations thereof; maintaining said culture in an initial growthphase under a first set of culture conditions for a first period of timesufficient to allow said cells to reproduce to a viable cell densitywithin a range of about 20%-80% of the maximal possible viable celldensity if said culture were maintained under the first set of cultureconditions; changing at least one of the culture conditions, so that asecond set of culture conditions is applied; maintaining said culturefor a second period of time under the second set of conditions and for asecond period of time so that the polypeptide accumulates in the cellculture.
 7. The method of claim 1, wherein said cell culture conditionin said changing at least one of the culture conditions step is selectedfrom the group consisting of: (i) temperature, (ii) pH, (iii)osmolality, (iv) chemical inductant level, and combinations thereof. 8.The method of claim 1, wherein the initial glutamine concentration ofsaid medium is less than or equal to 13 mM.
 9. The method of claim 1,wherein the initial glutamine concentration of said medium is less thanor equal to 10 mM.
 10. The method of claim 1, wherein the initialglutamine concentration of said medium is less than or equal to 7 mM.11. The method of claim 1, wherein the initial glutamine concentrationof said medium is less than or equal to 4 mM.
 12. The method of claim 1,wherein the total cumulative amount per unit volume of glutamine of saidmedium is less than or equal to 13 mM.
 13. The method of claim 1,wherein the total cumulative amount per unit volume of glutamine of saidmedium is less than or equal to 10 mM.
 14. The method of claim 1,wherein the total cumulative amount per unit volume of glutamine of saidmedium is less than or equal to 7 mM.
 15. The method of claim 1, whereinthe total cumulative amount per unit volume of glutamine of said mediumis less than or equal to 4 mM.
 16. The method of claim 1, whereinglutamine is only provided in the initial medium at the beginning of thecell culture.
 17. The method of claim 1, wherein the concentration ofsoluble iron in the media is greater than 5 μM.
 18. The method of claim1, wherein viable cell density of said culture is measured on a periodicbasis.
 19. The method of claim 1, wherein viability of said culture ismeasured on a periodic basis.
 20. The method of claim 1, wherein saidlactate levels of said culture is measured on a periodic basis.
 21. Themethod of claim 1, wherein said ammonium levels of said culture ismeasured on a periodic basis.
 22. The method of claim 1, wherein saidtiter of said culture is measured on a periodic basis.
 23. The method ofclaim 1, wherein osmolarity of said culture is measured on a periodicbasis.
 24. The method of claims 18-23, wherein said measurements aretaken daily.
 25. The method of claim 1, wherein the initial density ofsaid mammalian cells is at least 2×10² cells/mL.
 26. The method of claim1, wherein the initial density of said mammalian cells is at least 2×10³cells/mL.
 27. The method of claim 1, wherein the initial density of saidmammalian cells is at least 2×10⁴ cells/mL.
 28. The method of claim 1,wherein the initial density of said mammalian cells is at least 2×10⁵cells/mL.
 29. The method of claim 1, wherein the initial density of saidmammalian cells is at least 2×10⁶ cells/mL.
 30. The method of claim 1,wherein the initial density of said mammalian cells is at least 5×10⁶cells/mL.
 31. The method of claim 1, wherein the initial density of saidmammalian cells is at least 10×10⁶ cells/mL.
 32. The method of claim 1,wherein the step of providing comprises providing at least about 1000 Lof a culture.
 33. The method of claim 1, wherein the step of providingcomprises providing at least about 2500 L of a culture.
 34. The methodof claim 1, wherein the step of providing comprises providing at leastabout 5000 L of a culture.
 35. The method of claim 1, wherein the stepof providing comprises providing at least about 8000 L of a culture. 36.The method of claim 1, wherein the step of providing comprises providingat least about 10,000 L of a culture.
 37. The method of claim 1, whereinthe step of providing comprises providing at least about 12,000 L of aculture.
 38. The method of claim 1, wherein said first set of conditionscomprises a first temperature range that is approximately 30 to 42degrees Celsius.
 39. The method of claim 1, wherein said first set ofconditions comprises a first temperature range that is approximately 32to 40 degrees Celsius.
 40. The method of claim 1, wherein said first setof conditions comprises a first temperature range that is approximately34 to 38 degrees Celsius.
 41. The method of claim 1, wherein said firstset of conditions comprises a first temperature range that isapproximately 36 to 37 degrees Celsius.
 42. The method of claim 1,wherein said first set of conditions comprises a first temperature rangethat is approximately 37 degrees Celsius.
 43. The method of claim 1,wherein said second set of conditions comprises a second temperaturerange that is approximately 25 to 41 degrees Celsius.
 44. The method ofclaim 1, wherein said second set of conditions comprises a secondtemperature range that is approximately 27 to 38 degrees Celsius. 45.The method of claim 1, wherein said second set of conditions comprises asecond temperature range that is approximately 29 to 35 degrees Celsius.46. The method of claim 1, wherein said second set of conditionscomprises a second temperature range that is approximately 29 to 33degrees Celsius.
 47. The method of claim 1, wherein said second set ofconditions comprises a second temperature range that is approximately 30to 32 degrees Celsius.
 48. The method of claim 1, wherein said secondset of conditions comprises a second temperature range that isapproximately 31 degrees Celsius.
 49. The method of claim 1, furthercomprising a second changing step subsequent to first said changing atleast one of the culture conditions comprising changing at least one ofthe culture conditions, so that a third set of conditions is applied tothe culture.
 50. The method of claim 49, wherein the second changingstep comprises changing at least one culture condition selected from thegroup consisting of: (i) temperature, (ii) pH, (iii) osmolality, (iv)chemical inductant level, and combinations thereof.
 51. The method ofclaim 49, wherein said third set of conditions comprises a thirdtemperature range that is approximately 25 to 40 degrees Celsius. 52.The method of claim 49, wherein said third set of conditions comprises athird temperature range that is approximately 27 to 37 degrees Celsius.53. The method of claim 49, wherein said third set of conditionscomprises a third temperature range that is approximately 29 to 34degrees Celsius.
 54. The method of claim 49, wherein said third set ofconditions comprises a third temperature range that is approximately 30to 32 degrees Celsius.
 55. The method of claim 1, wherein said firstperiod of time is between 0-8 days.
 56. The method of claim 1, whereinsaid first period of time is between 1-7 days.
 57. The method of claim1, wherein said first period of time is between 2-6 days.
 58. The methodof claim 1, wherein said first period of time is between 3-5 days. 59.The method of claim 1, wherein said first period of time isapproximately 4 days.
 60. The method of claim 1, wherein said firstperiod of time is approximately 5 days.
 61. The method of claim 1,wherein said first period of time is approximately 6 days.
 62. Themethod of claim 1, wherein the total of said first period of time andsaid second period of time is at least 5 days.
 63. The method of claim1, wherein in the step of maintaining said culture for a second periodof time, the lactate level decreases subsequent to the lactate level inthe culture reaching a maximal level.
 64. The method of claim 1, whereinin the step of maintaining said culture for a second period of time, theammonium level decreases subsequent to the ammonium level in the culturereaching a maximal level.
 65. The method of claim 1, wherein said totalamount of said produced polypeptide is at least 1.5-fold higher that theamount of polypeptide produced under otherwise identical conditions inotherwise identical medium that lacks said medium characteristic. 66.The method of claim 1, wherein said total amount of said producedpolypeptide is at least 2-fold higher that the amount of polypeptideproduced under otherwise identical conditions in otherwise identicalmedium that lacks said medium characteristic.
 67. The method of claim 1,wherein said cell culture is further provided with supplementarycomponents.
 68. The method of claim 67, wherein said supplementarycomponents are provided at multiple intervals.
 69. The method of claim67 wherein said supplementary components are selected from a groupconsisting of hormones and/or other growth factors, particular ions(such as sodium, chloride, calcium, magnesium, and phosphate), buffers,vitamins, nucleosides or nucleotides, trace elements (inorganiccompounds usually present at very low final concentrations), aminoacids, lipids, or glucose or other energy source.
 70. A method ofproducing a polypeptide in a large-scale production cell culturecomprising steps of; providing a cell culture comprising; mammaliancells that contain a gene encoding a polypeptide of interest, which geneis expressed under condition of cell culture; and a defined mediumcontaining glutamine and having at least two medium characteristicsselected from the group consisting of: i) a starting amino acidconcentration greater than about 70 mM, ii) a molar glutamine toasparagine ratio of less than about 2, iii) a molar glutamine to totalamino acid ratio of less than about 0.2, iv) a molar inorganic ion tototal amino acid ratio between about 0.4 to 1, and v) a combinedglutamine and asparagine concentration greater than about 16 mM;maintaining said culture in an initial growth phase under a first set ofculture conditions for a first period of time sufficient to allow saidcells to reproduce within a range of about 20%-80% of the maximalpossible viable cell density if said culture were maintained under thefirst set of culture conditions; changing at least one of the cultureconditions, so that a second set of culture conditions is applied;maintaining said culture for a second period of time under the secondset of conditions and for a second period of time so that thepolypeptide accumulates in the cell culture.
 71. A method of producing apolypeptide in a large-scale production cell culture comprising stepsof; providing a cell culture comprising; mammalian cells that contain agene encoding a polypeptide of interest, which gene is expressed undercondition of cell culture; and a defined medium containing glutamine andhaving at least three medium characteristic selected from the groupconsisting of: i) a starting amino acid concentration greater than about70 mM, ii) a molar glutamine to asparagine ratio of less than about 2,iii) a molar glutamine to total amino acid ratio of less than about 0.2,iv) a molar inorganic ion to total amino acid ratio between about 0.4 to1, and v) a combined glutamine and asparagine concentration greater thanabout 16 mM; maintaining said culture in an initial growth phase under afirst set of culture conditions for a first period of time sufficient toallow said cells to reproduce within a range of about 20%-80% of themaximal possible viable cell density if said culture were maintainedunder the first set of culture conditions; changing at least one of theculture conditions, so that a second set of culture conditions isapplied; maintaining said culture for a second period of time under thesecond set of conditions and for a second period of time so that thepolypeptide accumulates in the cell culture.
 72. A method of producing apolypeptide in a large-scale production cell culture comprising stepsof; providing a cell culture comprising; mammalian cells that contain agene encoding a polypeptide of interest, which gene is expressed undercondition of cell culture; and a defined medium containing glutamine andhaving at least four medium characteristic selected from the groupconsisting of: i) a starting amino acid concentration greater than about70 mM, ii) a molar glutamine to asparagine ratio of less than about 2,iii) a molar glutamine to total amino acid ratio of less than about 0.2,iv) a molar inorganic ion to total amino acid ratio between about 0.4 to1, and v) a combined glutamine and asparagine concentration greater thanabout 16 mM; maintaining said culture in an initial growth phase under afirst set of culture conditions for a first period of time sufficient toallow said cells to reproduce within a range of about 20%-80% of themaximal possible viable cell density if said culture were maintainedunder the first set of culture conditions; changing at least one of theculture conditions, so that a second set of culture conditions isapplied; maintaining said culture for a second period of time under thesecond set of conditions and for a second period of time so that thepolypeptide accumulates in the cell culture.
 73. A method of producing apolypeptide in a large-scale production cell culture comprising stepsof; providing a cell culture comprising; mammalian cells that contain agene encoding a polypeptide of interest, which gene is expressed undercondition of cell culture; and a defined medium containing glutamine,characterized by: i) a starting amino acid concentration greater thanabout 70 mM, ii) a molar glutamine to asparagine ratio of less thanabout 2, iii) a molar glutamine to total amino acid ratio of less thanabout 0.2, iv) a molar inorganic ion to total amino acid ratio betweenabout 0.4 to 1, and v) a combined glutamine and asparagine concentrationgreater than about 16 mM; maintaining said culture in an initial growthphase under a first set of culture conditions for a first period of timesufficient to allow said cells to reproduce within a range of about20%-80% of the maximal possible viable cell density if said culture weremaintained under the first set of culture conditions; changing at leastone of the culture conditions, so that a second set of cultureconditions is applied; maintaining said culture for a second period oftime under the second set of conditions and for a second period of timeso that the polypeptide accumulates in the cell culture.
 74. A method ofproducing a polypeptide in a large-scale production cell culturecomprising the steps of: providing a cell culture comprising; mammaliancells that contain a gene encoding a polypeptide of interest, which geneis expressed under condition of cell culture; and a medium containingglutamine and having a combined cumulative amount of glutamine andasparagine per unit volume of greater than about 16 mM; maintaining saidculture in an initial growth phase under a first set of cultureconditions for a first period of time sufficient to allow said cells toreproduce within a range of about 20%-80% of the maximal possible viablecell density if said culture were maintained under the first set ofculture conditions; changing at least one of the culture conditions, sothat a second set of culture conditions is applied; maintaining saidculture for a second period of time under the second set of conditionsand for a second period of time so that that the polypeptide accumulatesin the cell culture.
 75. The method of claim 1, wherein said mediumcomprises a medium containing glutamine and having a mediumcharacteristic selected from the group consisting of: (i) a startingamino acid concentration greater than about 70 mM, (ii) a molar startingglutamine to starting asparagine ratio of less than about 2, (iii) amolar starting glutamine to starting total amino acid ratio of less thanabout 0.2, (iv) a molar starting inorganic ion to starting total aminoacid ratio between about 0.4 to 1, (v) a combined starting glutamine andstarting asparagine concentration greater than about 16 mM, andcombinations thereof.
 76. The method of any one of claims 1-6 or 70-75,wherein: lactate levels are lower than those levels observed underotherwise identical conditions in otherwise identical medium that lackssaid medium characteristic; ammonium levels are lower than those levelsobserved under otherwise identical conditions in otherwise identicalmedium that lacks said medium characteristic; and total amount ofproduced polypeptide is at least as high as that observed underotherwise identical conditions in otherwise identical medium that lackssaid medium characteristic.
 77. The method of claim 1, wherein saidculture is not supplemented with additional components over the courseof producing said polypeptide.
 78. The method of claim 1, wherein saidculture is not supplemented with additional glutamine over the course ofproducing said polypeptide.
 79. The method of claim 1, wherein theglutamine concentration in said culture is substantially depleted priorto said step of changing to a second set of culture conditions.
 80. Themethod of claim 1, wherein the glutamine concentration in said cultureis substantially depleted at approximately the same time as said step ofchanging to a second set of culture conditions.
 81. The method of claim1, wherein glycylglutamine is substituted for glutamine in said culture.82. The method of claim 1, wherein said medium contains: (i) acumulative amino acid amount per unit volume greater than about 70 mM,(ii) a molar cumulative glutamine to cumulative asparagine ratio of lessthan about 2, (iii) a molar cumulative glutamine to cumulative totalamino acid ratio of less than about 0.2, (iv) a molar cumulativeinorganic ion to cumulative total amino acid ratio between about 0.4 to1, and (v) a combined cumulative amount of glutamine and asparagine perunit volume greater than about 16 mM.
 83. The method of claim 1, whereinsaid medium contains: (i) a cumulative amino acid amount per unit volumegreater than about 70 mM, (ii) a molar cumulative glutamine tocumulative total amino acid ratio of less than about 0.2, (iii) a molarcumulative inorganic ion to cumulative total amino acid ratio betweenabout 0.4 to 1, and (iv) a combined cumulative amount of glutamine andasparagine per unit volume greater than about 16 a mM.
 84. The method ofclaim 1, wherein the cumulative total amount of histidine, isoleucine,leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, andproline per unit volume in said medium is greater than approximately 25mM.
 85. The method of claim 1, wherein the cumulative total amount ofhistidine, isoleucine, leucine, methionine, phenylalanine, proline,tryptophan, tyrosine, and proline per unit volume in said medium isgreater than approximately 35 mM.
 86. The method of claim 1, wherein theinitial total amount of histidine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine, and proline per unitvolume in said medium is greater than approximately 25 mM.
 87. Themethod of claim 1, wherein the initial total amount of histidine,isoleucine, leucine, methionine, phenylalanine, proline, tryptophan,tyrosine, and proline per unit volume in said medium is greater thanapproximately 35 mM.
 88. The method of claim 1, wherein said medium hasa medium characteristic selected from the group consisting of: (i) acumulative total amount of histidine per unit volume greater thanapproximately 1.7 mM; (ii) a cumulative total amount of isoleucine perunit volume greater than approximately 3.5 mM; (iii) a cumulative totalamount of leucine per unit volume greater than approximately 5.5 mM;(iv) a cumulative total amount of methionine per unit volume greaterthan approximately 2.0 mM; (v) a cumulative total amount ofphenylalanine per unit volume greater than approximately 2.5 mM; (vi) acumulative total amount of proline per unit volume greater thanapproximately 2.5 mM; (vii) a cumulative total amount of tryptophan perunit volume greater than approximately 1.0 mM; (viii) a cumulative totalamount of tyrosine per unit volume greater than approximately 2.0 mM;and (ix) a cumulative total amount of proline per unit volume greaterthan approximately 2.5 mM.
 89. The method of claim 1, wherein saidmedium has a medium characteristic selected from the group consistingof: (i) an initial amount of histidine per unit volume greater thanapproximately 1.7 mM; (ii) an initial amount of isoleucine per unitvolume greater than approximately 3.5 mM; (iii) an initial amount ofleucine per unit volume greater than approximately 5.5 mM; (iv) aninitial amount of methionine per unit volume greater than approximately2.0 mM; (v) an initial amount of phenylalanine per unit volume greaterthan approximately 2.5 mM; (vi) an initial amount of proline per unitvolume reater than approximately 2.5 mM; (vii) an initial amount oftryptophan per unit volume greater than approximately 1.0 mM; (viii) aninitial amount of tyrosine per unit volume greater than approximately2.0 mM; and (ix) an initial amount of proline per unit volume greaterthan approximately 2.5 mM.
 90. The method of claim 1, wherein thecumulative total amount of serine per unit volume in said medium isgreater than approximately 7 mM.
 91. The method of claim 1, wherein thecumulative total amount of serine per unit volume in said medium isgreater than approximately 10 mM.
 92. The method of claim 1, wherein thecumulative total amount of asparagine per unit volume in said medium isgreater than approximately 8 mM.
 93. The method of claim 1, wherein thecumulative total amount of asparagine per unit volume in said medium isgreater than approximately 12 mM.
 94. The method of claim 1, wherein theinitial total amount of asparagine per unit volume in said medium isgreater than approximately 8 mM.
 95. The method of claim 1, wherein theinitial total amount of asparagine per unit volume in said medium isgreater than approximately 12 mM.
 96. The method of claim 1, wherein thecumulative total amount of phosphorous per unit volume in said medium isgreater than approximately 2.5 mM.
 97. The method of claim 1, whereinthe cumulative total amount of phosphorous per unit volume in saidmedium is greater than approximately 5 mM.
 98. The method of claim 1,wherein the cumulative total amount of glutamate per unit volume in saidmedium is less than approximately 1 mM.
 99. The method of claim 1,wherein the cumulative total amount of calcium pantothenate per unitvolume in said medium is greater than approximately 8 mg/L.
 100. Themethod of claim 1, wherein the cumulative total amount of calciumpantothenate per unit volume in said medium is greater thanapproximately 20 mg/L.
 101. The method of claim 1, wherein thecumulative total amount of nicotinamide per unit volume in said mediumis greater than approximately 7 mg/L.
 102. The method of claim 1,wherein the cumulative total amount of nicotinamide per unit volume insaid medium is greater than approximately 25 mg/L.
 103. The method ofclaim 1, wherein the cumulative total amount of pyridoxine and pyridoxalper unit volume in said medium is greater than approximately 5 mg/L.104. The method of claim 1, wherein the cumulative total amount ofpyridoxine and pyridoxal per unit volume in said medium is greater thanapproximately 35 mg/L.
 105. The method of claim 1, wherein thecumulative total amount of riboflavin per unit volume in said medium isgreater than approximately 1.0 mg/L.
 106. The method of claim 1, whereinthe cumulative total amount of riboflavin per unit volume in said mediumis greater than approximately 2.0 mg/L.
 107. The method of claim 1,wherein the cumulative total amount of thiamine hydrochloride per unitvolume in said medium is greater than approximately 7 mg/L.
 108. Themethod of claim 1, wherein the cumulative total amount of thiaminehydrochloride per unit volume in said medium is greater thanapproximately 35 mg/L.
 109. The method of claim 1, wherein thepolypeptide is anti-GDF-8.
 110. The method of claim 1, wherein thepolypeptide is anti-LewY. 111-114. (canceled)